Multivalent polymyxin antibiotics

ABSTRACT

Disclosed are multibinding compounds which are antibiotics effective against bacterial infections, in particular, Gram-negative bacterial infections. The multibinding compounds of this invention containing from 2 to 10 ligands covalently attached to one or more linkers. Each ligand is a polymyxin, circulin or octapeptin antibiotic or other suitable compound which binds to the LPS present in bacteria, in particular, Gram-negative bacteria. The multibinding compounds of this invention are useful for the prophylaxis and treatment of various bacterial infections caused by bacteria.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel multibinding compounds (agents) that are useful as antibiotics, and to pharmaceutical compositions comprising such compounds. The compounds are useful medications for the prophylaxis and treatment of various bacterial infections.

REFERENCES

The following publications are cited in this application:

Storm et al., “Polymyxin and related peptide antibiotics,” Ann. Rev. Biochem., 46: 723-763 (1977).

Weinstein et al., “Selective chemical modifications of Polymyxin B,” Bioorganic and Medicinal Chemistry Letters, 8: 3391-3396 (1998).

Kimura and Matsunaga, “Polymyxin B octapeptin and polymyxin B heptapeptide are potent outer membrane permeability-increasing agents,” Journal of Antibiotics, 45(5): 742-749 (1992).

Srinivasa and Ramachandran, “Chemical modification of peptide antibiotics: Part VI-Biological activity of derivatives of polymyxin B,” Indian J. Biochemistry and Biophysics, 14: 54-58 (1978).

PCT WO 88/00950 to Fauchere et al.

DE Patent No. 1,906,699 to Pfizer.

DE Patent No. 2,204,887 to Rhone-Poulenc.

J. E. Kapusnik-Uner, M. A. Sande, H. F. Chambers in Goodman & Gilman's “The Pharmacological Basis of Therapeutics,” 9h Ed. (J. G. Hardman, L. E. Limbird, P. B. Molinoff, R. W. Ruddon, A. G. Gilman, Eds.); McGraw-1-Ell, New York: p 1123-1153 (1996).

M. R. W. Brown, S. M Wood, J. Pharm. Pharmacol. 24: 215-218 (1972).

S. Srimal, N. Surolia, S. Balasubramanian, A. Surolia, Biochem. J., 315: 679-686 (1996).

J. L. Shenep, R. P. Barton, et al., J. Infect. Dis. 151: 1012-1018 (1984).

M. G. Tauber, A. M. Shibl, C. J. Hackbarth, J. W. Larrick, M. A. Sande, Antimicrob. Agents Chemother. 156: 456-462 (1987).

G. S. Doig, C. M. Martin, et al., Crit. Care Med. 25:1956-1961 (1997).

C. Verwaest, J. Verhaegen, P. Ferdinande, M. Schetz, G. Van Den Berghe, L. Verbist, P. Lauwers, Crit. Care Med. 25: 63-71 (1997).

G. S. Bauldoff, D. R. Nunley, J. D. Manzetti, J. H. Dauber, R. J. Keenan, Transplantation 64: 748-752 (1997).

P. Diot, F. Gangadoux, C. Martin, H. Ellataoui, Y. Furet, M. Breteau, E. Boissinot, E. Lemarie, Eur. Resp. J. 10: 1995-1998 (1997).

S. E. Bucklin, P. Lake, L. Logdberg, D. C. Morrison, Antimicrobial Agents Chemother. 39: 1462-1466 (1995).

B. L. Jaber, B. J. Pereira, Am. J. Kidney Dis. 30(Suppl. 4): S44-56 (1997).

J. R. Berg, C. M. Spilker, S. A. Lewis, J. Membrane Biol. 154: 119-130 (1996).

L. Weinstein in “The Pharmacological Basis of Therapeutics,” 5 1h Ed. (L. S. Goodman, A. Gilman, Eds.); MacMillan, N.Y.:, p1230-1233 (1975).

Physician's Desk Reference, Medical Economics Co., Oradell, N.J.: (1993).

M. Helander, Y. Kato, I. Kilpelainen, R. Kostiainen, B. Lindner, K. Nummila, T. Sugiyama, T. Yokochi, Eur. J. Biochem. 237: 272-278 (1996).

Entries 2542 and 7734, The Merck Index, 12 th Edition, Merck and Co., Whitehouse Station, N.J.: (1996).

K. Vogler, R. O. Studer, W. Lergier, P. Lanz, Helv. Chim. Acta 43: 1751-1760 (1960).

K. Vogler, R. O. Studer, P. Lanz, W. Lergier, E. Boehni, Experientia 20: 365-366 (1964).

T. Kurihara, H. Takeda, H. Ito, Yakugaku Zasshi 92: 129-134 (1972).

K. Nakajima, Chem. Pharm. Bull. 15: 1219-1224 (1967).

M. Teuber, Z. Naturforsch. Teil B 25: 117 (1970).

Y. Kimura, H. Matsunaga, M. Vaara, J. Antibiot. 45: 742-749 (1992).

S. Chihara, A. Ito, M Yahata, T. Tobita, Y. Koyama, Agric. Biol. Chem. 38: 521-529 (1974).

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D. A. Stoma, K. S. Rosenthal, P. E. Swanson, Ann. Rev. Biochem. 46: 723-763 (1977).

M. Vaara, Drugs Exp. Clin. Res. 17: 437-444 (1991).

PCT WO 90/15628.

G. Radhakrishna, L. K. Ramachandran, Indian J. Biochem. Biophys. 20: 213-217 (1983).

C. P. Coyne, J. T. Moritz, J. Endotoxin Res. 1: 207-215 (1994).

J. L. Fauchere, K. Mosbach CH Appl. 86/315106 August 1986.

All of the above publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

2. State of the Art

Bacteria are ubiquitous microbes capable of causing significant morbidity and mortality in infected individuals. Healthy individuals, having intact immune systems, rapidly eliminate pathogenic bacteria. However, many conditions render patients vulnerable to bacterial infection. Thus, individuals suffering from primary immunodeficiency disorders, such as AIDS, commonly develop infections. Alternatively, individuals may become susceptible to bacterial infection as the result of secondary immunodeficiencies due to other underlying disorders. For example, patients with diseases such as diabetes, connective tissue disorders, or trauma frequently develop complications due to severe bacterial infections. In such patients, overwhelming bacterial infections may result in a cascade of physiological changes leading to septic (or endotoxic) shock, which often culminates in the patient mortality.

Although most bacteria are capable of producing sepsis, a sub-class of bacteria, known as Gram-negative bacteria, which includes Eschericia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa are the usual etiologic agents. The profound pathogenic effects of these microbes are due to a structural component, unique to Gram-negative bacteria, known as an outer membrane.

The outer membrane, which surrounds the bacterial cell, and protects it from environmental assaults, includes a molecule known as lipopolysaccharide (LPS). LPS is a complex structure with three components: 1) an outer region consisting of polymerized di- to penta-saccharide repeating units whose composition varies with bacterial species; 2) an inner region including of oligosaccharides linked by a sugar 2-keto-3-deoxy-C-mannose-octonate to a disaccharide backbone; and lipid A. This latter molecule, a glucosamine disaccharide with attached phosphate and acyl (fatty acid) groups, is responsible for most of the biological activity of the molecule.

The pathological effects of LPS are due to both intact LPS present in the outer membrane of the cell (bound LPS) and LPS that is released from the membrane and shed into blood (soluble LPS). Regardless of form, LPS elicits its biological effects by binding to a receptor found on a mononuclear phagocyte known as a monocyte. The interaction stimulates cellular processes resulting in the release of pro-inflammatory mediators such as TNF-α, IL-1β, IL-6 and PGE₂, which, then leads to arterial hypotension, metabolic acidosis, decreased systemic vascular resistance, tachypnea and organ dysfunction that characterize septic shock.

Bacterial infections are usually treated with a molecularly diverse group of agents known as antibiotics, which act by a wide variety of mechanisms well known to those skilled in the art. While these drugs are sometimes capable of resolving the effects of bacteremia, infections with Gram-negative bacteremia presents special challenges. For example, treatment with conventional antibiotics while leading to the death of the pathogen, results in the release of toxic bacterial components, such as LPS. Thus, treatment with antibiotics may increase the amount of LPS or products of LPS such as endotoxin into the circulation.

Certain antibiotics, however, are able to neutralize the action of LPS, and mitigate its effects by binding to the molecule. Examples of such antibiotics include the polymyxin, circulin and octapeptin antibiotics, most notably polymyxin B and polymyxin E (also known as colistin), which are cyclic polypeptide compounds produced by strains of Bacillus polymyxa.

The lipid-bearing, polycationic polymyxin forms a complex with anionic phospholipids of the LPS and inserts into the membrane. This event disrupts the LPS, leading to loss of essential intracellular components and rapid bacterial cell death. In addition to its killing effects on intact bacteria, polymyxin also has high affinity for “free” LPS components, most importantly, the lipid A portion of the lipopolysaccharide of the LPS (endotoxin). Complexation of lipid A by polymyxin prevents most of the pathophysiologic consequences of endotoxin in experimental systems.

Combinations of polymyxin B sulfate and/or colistin sulfate with various other compounds are widely used in opthalmic, otic, and topical applications against Gram-negative organisms. Strains of Enterobacter, E. coli, Klebsiella, Salmonella, Pasteurella, Bordetella, and Shigella are typically sensitive to polymyxins at concentrations of 0.05-2.0 micrograms/mL in vitro, while most strains of Pseudomonas aeruginosa are inhibited by less than 8 micrograms/mL. Intrinsically resistant strains include Proteus mirabilis, Serratia marcesens, Providencia, and Edwardsiella tarda. More recently introduced uses of the polymyxins include the use of oral colistin for prophylactic gut clearance and of nebulized colistin for treatment of Pseudomonas infections in cystic fibrosis patients. In current development are the systemic use of macromolecular polymyxin-dextran conjugates and the extracorporeal use of polymyxin adsorbents for intervention in Gram-negative sepsis.

Polymyxin was at an earlier time administered parenterally and colistin sulfate is formulated for parenteral use; however, parenteral use of polymyxins is rare due to their nephrotoxicity and neurotoxicity. These effects are believed to have as their source the interaction of the polymyxins with phospholipids of mammalian cells. Neurological side effects of colistin administration include circumoral parasthesia pain at the site of intramuscular injection, numbness, tingling, or formication in the extremities, generalized pruritis, vertigo, dizziness, slurring of speech, and respiratory paralysis via neuromuscular blockade. Nephrological side effects include acute tubular necrosis, interstitial nephritis, proteinuria, hematuria, cylindruria, azotemia, and reduced glomerular filtration rate. The magnitude of these effects increases with continued therapy; however, the effects are generally reversible upon cessation of treatment.

For these reasons, polymyxin B, in intravenous form, is only given to hospitalized patients under constant supervision. Polymyxins and related antibiotics are not used routinely for systemic infections. Application of polymyxins to intact skin, denuded skin, or mucous membranes results in no systemic reactions because the drugs are poorly absorbed. Side effects following large (600 mg) oral doses of the antibiotic include nausea, vomiting, and diarrhea. Neurotoxic reactions have additionally been observed, the most severe being respiratory paralysis when given soon after anesthesia and/or muscle relaxants.

Given polymyxin's significant systemic toxicities, the advent of anti-pseudomonal β-lactams, the fluoroquinolone family of antibacterial agents, and aminoglycosides effective against Gram-negative organisms superseded parenteral use of polymyxin.

Antibacterial agents are important weapons in the fight against pathogenic bacteria. However, an increasing problem with respect to the effectiveness of antibacterial agents relates to the emergence of strains of bacteria that are highly resistant to such agents. Microbial resistance to the polymyxins is slow to develop and typically involves alterations in the composition of LPS components.

It would therefore be desirable to find antibacterial agents that are active against Gram-negative bacteria, in particular, drug resistant strains. It would also be advantageous to have antibacterial agents that demonstrate high activity and selectivity toward their targets and have high bioavailability, low toxicity, for example, nephrotoxicity, and other side effects. The present invention provides such agents.

SUMMARY OF THE INVENTION

This invention is directed to novel multibinding polymyxin, circulin and octapeptin antibiotics. The multibinding compounds of this invention are useful in the treatment and prevention of bacterial infections.

In particular, the compounds can be used to treat bacterial infections caused by Gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, Enterobacter sp., Salmonella sp., Pasteurella sp., Bordetella sp., Shigella sp., Proteus mirabilis, and Serratia marcesens.

In one of its composition aspects, this invention provides a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a polymyxin, circulin or octapeptin antibiotic or other suitable compound which binds to the LPS present in bacteria, in particular, Gram-negative bacteria.

In another of its composition aspects, this invention provides a multibinding compound of formula I:

(L)_(p)(X)_(q)  I

wherein each L is independently a ligand comprising a polymyxin, circulin or octapeptin antibiotic or other suitable compound which exhibits multibinding properties toward LPS and/or which demonstrates antibacterial properties; each X is independently a linker; p is an integer of from 2 to 10; and q is an integer of from 1 to 20; and pharmaceutically-acceptable salts thereof.

Preferably, q is less than p in the multibinding compounds of this invention.

Examples of suitable ligands include polymyxin A, polymyxin B1, polymyxin B2, polymyxin D1, polymyxin E1, polymyxin E2, circulin A, octapeptin A1, octapeptin A2, octapeptin A3, octapeptin B1, octapeptin B2, octapeptin B3, octapeptin C1. The γ-amino group on the DAB subunits on the ligands is a preferred site for attachment to the linker(s).

In still another of its composition aspects, this invention provides a multibinding compound of formula II:

L′—X′—L′  II

wherein each L′ is independently a ligand comprising a polymyxin, circulin or octapeptin antibiotic or other suitable compound which exhibits multibinding properties toward LPS and/or which demonstrates antibacterial properties; and X′ is a linker; and pharmaceutically-acceptable salts thereof.

Preferably, in the above embodiments, each linker (i.e., X, X′ or X″) independently has the formula:

—X^(a)—Z—(Y^(a)—Z)_(m)—Y^(b)—Z—X^(a)—

wherein

m is an integer of from 0 to 20;

X^(a) at each separate occurrence is selected from the group consisting of —O—, —S—, —NR—, —C(O)—, —C(O)O—, —C(O)NR—, —C(S), —C(S)O—, —C(S)NR— or a covalent bond where R is as defined below;

Z is at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond;

Y^(a) and Y^(b) at each separate occurrence are selected from the group consisting of —C(O)NR′—, —NR′C(O)—, —NR′C(O)NR′—, —C(═NR′)—NR′—, —NR′—C(═NR′)—, —NR′—C(O)—O—, —N═C(X^(a))—NR′—, —P(O)(OR′)—O—, —S(O)_(n)CR′R″—, —S(O)_(n)—NR′—, —S—S— and a covalent bond; where n is 0, 1 or 2; and R, R′ and R″ at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic.

In yet another of its composition aspects, this invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a polymyxin, circulin or octapeptin antibiotic or other suitable compound which exhibits multibinding properties toward LPS present in certain bacteria, especially Gram-negative bacteria; and/or which demonstrates antibacterial properties; and pharmaceutically-acceptable salts thereof.

This invention is also directed to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound of formula I or II.

The multibinding compounds of this invention are effective antibiotics, which are useful for treating a variety of bacterial infections. Accordingly, in one of its method aspects, this invention provides a method for treating various bacterial infections. Examples of such infections include infections caused by Enterobacter sp., E. coli, Klebsiella sp., Salmonella sp., Pasteurella sp., Bordetella sp., Shigella sp., Pseudomonas aeruginosa, Proteus mirabilis, and Serratia marcesens.

When used to treat bacterial infections, for example, the method involves administering to a patient having a bacterial infection a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a therapeutically-effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a polymyxin, circulin or octapeptin antibiotic or other suitable compound which exhibits multibinding properties toward LPS present in certain bacteria, especially Gram-negative bacteria; and/or which demonstrates antibacterial properties; and pharmaceutically-acceptable salts thereof.

This invention is also directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties with respect to various sites on bacteria. The diverse multimeric compound libraries provided by this invention are synthesized by combining a linker or linkers with a ligand or ligands to provide for a library of multimeric compounds wherein the linker and ligand each have complementary functional groups permitting covalent linkage. The library of linkers is preferably selected to have diverse properties such as valency, linker length, linker geometry and rigidity, hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity and polarizability and/or polarization. The library of ligands is preferably selected to have diverse attachment points on the same ligand, different functional groups at the same site of otherwise the same ligand, and the like.

This invention is also directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties with respect to sites on various bacteria. The diverse multimeric compound libraries provided by this invention are synthesized by combining a linker or linkers with a ligand or ligands to provide for a library of multimeric compounds wherein the linker and ligand each have complementary functional groups permitting covalent linkage. The library of linkers is preferably selected to have diverse properties such as valency, linker length, linker geometry and rigidity, hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity and polarizability and/or polarization. The library of ligands is preferably selected to have diverse attachment points on the same ligand, different functional groups at the same site of otherwise the same ligand, and the like.

This invention is also directed to libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties with respect to various sites on bacteria. These libraries are prepared via the methods described above and permit the rapid and efficient evaluation of what molecular constraints impart multibinding properties to a ligand or a class of ligands which bind to the LPS present in bacteria, especially gram negative bacteria, and/or which demonstrate antibacterial properties.

Accordingly, in one of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds which exhibit multibinding properties toward LPS and/or which demonstrate antibacterial properties, which method comprises:

(a) identifying a ligand or a mixture of ligands which includes a polymyxin, circulin or octapeptin compound or other suitable compound which binds to the LPS present in bacteria and/or which demonstrates antibacterial properties, wherein each ligand contains at least one reactive functionality;

(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;

(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and

(d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties.

In another of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds which exhibit multibinding properties toward LPS and/or which demonstrate antibacterial properties, which method comprises:

(a) identifying a library of ligands which includes polymyxin, circulin or octapeptin and other suitable compounds which bind to the LPS present in bacteria and/or which demonstrate antibacterial properties wherein each ligand contains at least one reactive functionality;

(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;

(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and

(d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties.

The preparation of the multimeric ligand compound library is achieved by either the sequential or concurrent combination of the two or more stoichiometric equivalents of the ligands identified in (a) with the linkers identified in (b). Sequential addition is preferred when a mixture of different ligands is employed to ensure heteromeric or multimeric compounds are prepared. Concurrent addition of the ligands occurs when at least a portion of the multimer compounds prepared are homomultimeric compounds.

The assay protocols recited in (d) can be conducted on the multimeric ligand compound library produced in (c) above, or preferably, each member of the library is isolated by preparative liquid chromatography mass spectrometry (LCMS).

In one of its composition aspects, this invention is directed to a library of multimeric ligand compounds which exhibit multibinding properties toward LPS and/or which demonstrate antibacterial properties which library is prepared by the method comprising:

(a) identifying a ligand or a mixture of ligands which include polymyxin, circulin or octapeptin compounds or other suitable compounds which bind to the LPS present in certain bacteria, especially Gram-negative bacteria; and/or which demonstrate antibacterial properties; wherein each ligand contains at least one reactive functionality;

(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and

(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.

In another of its composition aspects, this invention is directed to a library of multimeric ligand compounds which exhibit multibinding properties toward LPS and/or which demonstrate antibacterial properties and which may possess multivalent properties which library is prepared by the method comprising:

(a) identifying a library of ligands which includes a polymyxin, circulin or octapeptin compound or other suitable compounds which bind to the LPS present in bacteria and/or which demonstrate antibacterial properties wherein each ligand contains at least one reactive functionality;

(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and

(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.

In a preferred embodiment, the library of linkers employed in either the methods or the library aspects of this invention is selected from the group comprising flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers of different geometry, acidic linkers, basic linkers, linkers of different polarizability and/or polarization and amphiphilic linkers. For example, in one embodiment, each of the linkers in the linker library may comprise linkers of different chain length and/or having different complementary reactive groups. Such linker lengths can preferably range from about 2 to 100 Å.

In another preferred embodiment, the ligand or mixture of ligands is selected to have reactive functionality at different sites on the ligands in order to provide for a range of orientations of said ligand on said multimeric ligand compounds. Such reactive functionality includes, by way of example, carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, pseudohalides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates and precursors thereof. It is understood, of course, that the reactive functionality on the ligand is selected to be complementary to at least one of the reactive groups on the linker so that a covalent linkage can be formed between the linker and the ligand.

In other embodiments, the multimeric ligand compound is homomeric (i.e., each of the ligands is the same, although it may be attached at different points) or heteromeric (i.e., at least one of the ligands is different from the other ligands).

In addition to the combinatorial methods described herein, this invention provides for an iterative process for rationally evaluating what molecular constraints impart multibinding properties to a class of antibacterial multimeric compounds or ligands. Specifically, this method aspect is directed to a method for identifying multimeric ligand compounds possessing multibinding properties with respect to the LPS present in certain bacteria, especially Gram-negative bacteria, or which possess antibacterial properties, which method comprises:

(a) preparing a first collection or iteration of multimeric compounds which is prepared by contacting at least two stoichiometric equivalents of the ligand or mixture of ligands which bind to the LPS present in certain bacteria, especially Gram-negative bacteria; and/or which demonstrate antibacterial properties; with a linker or mixture of linkers wherein said ligand or mixture of ligands comprises at least one reactive functionality and said linker or mixture of linkers comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand wherein said contacting is conducted under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands;

(b) assaying said first collection or iteration of multimeric compounds to assess which if any of said multimeric compounds possess multibinding properties;

(c) repeating the process of (a) and (b) above until at least one multimeric compound is found to possess multibinding properties;

(d) evaluating what molecular constraints imparted multibinding properties to the multimeric compound or compounds found in the first iteration recited in (a)-(c) above;

(e) creating a second collection or iteration of multimeric compounds which elaborates upon the particular molecular constraints imparting multibinding properties to the multimeric compound or compounds found in said first iteration;

(f) evaluating what molecular constraints imparted enhanced multibinding properties to the multimeric compound or compounds found in the second collection or iteration recited in (e) above;

(g) optionally repeating steps (e) and (f) to further elaborate upon said molecular constraints.

Preferably, steps (e) and (f) are repeated at least two times, more preferably at from 2-50 times, even more preferably from 3 to 50 times, and still more preferably at least 5-50 times.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of Polymyxin B₁ and B₂.

FIG. 2 is a representative structure of Polymyxin B which will be used in the schemes and examples.

FIG. 3 represents the synthesis of the 15 possible orientations of Polymyxin B dimers linked via DAB amino acids moeties of Polymyxin B. (Scheme A).

FIG. 4 represents the selective protection of the amine groups of Polymyxin B. (Scheme B).

FIG. 5 represents the synthesis of bis(DAB₁,DAB₁-polymyxin B) dimer. (Scheme C).

FIG. 6 represents the synthesis of bis(DAB₉,DAB₉-polymyxin B) dimer. (Scheme D).

FIG. 7 represents the synthesis of bis(DAB₁,DAB₉-polymyxin B) dimer. (Scheme E).

***Note: Although only Polymyxin B₁ and B₂ are shown in the figures and examples, the same reactions can be performed on the DAB units of other polymyxin, circulin and octapeptin ligands described herein, for example, Polymyxin A, D₁, D₂, E₁ (Colistin A) and E₂ (Colistin B).

FIG. 8 illustrates examples of multibinding compounds comprising 2 ligands attached in different forms to a linker.

FIG. 9 illustrates examples of multibinding compounds comprising 3 ligands attached in different forms to a linker.

FIG. 10 illustrates examples of multibinding compounds comprising 4 ligands attached in different forms to a linker.

FIG. 11 illustrates examples of multibinding compounds comprising 5-10 ligands attached in different forms to a linker.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to multibinding compounds which are useful as antibiotics, pharmaceutical compositions containing such compounds and methods for treating bacterial infections. When discussing such compounds, compositions or methods, the following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

The term “alkyl” refers to a monoradical branched or unbranched saturated hydrocarbon chain preferably having from 1 to 40 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, n-decyl, tetradecyl, and the like.

The term “substituted alkyl” refers to an alkyl group as defined above, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “alkylene” refers to a diradical of a branched or unbranched saturated hydrocarbon chain, preferably having from 1 to 40 carbon atoms, more preferably 1 to 10 carbon atoms and even more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—), the propylene isomers (e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

The term “substituted alkylene” refers to an alkylene group, as defined above, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Additionally, such substituted alkylene groups include those where 2 substituents on the alkylene group are fused to form one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fused to the alkylene group. Preferably such fused groups contain from 1 to 3 fused ring structures.

The term “alkaryl” refers to the groups -alkylene-aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein. Such alkaryl groups are exemplified by benzyl, phenethyl and the like.

The term “alkoxy” refers to the groups alkyl-O—, alkenyl-O—, cycloalkyl-O—, cycloalkenyl-O—, and alkynyl-O—, where alkyl, alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein. Preferred alkoxy groups are alkyl-O— and include, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—, substituted alkenyl-O—, substituted cycloalkyl-O—, substituted cycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl and substituted alkylene-O-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein. Preferred alkylalkoxy groups are alkylene-O-alkyl and include, by way of example, methylenemethoxy (—CH₂OCH₃), ethylenemethoxy (—CH₂CH₂OCH₃), n-propylene-iso-propoxy (—CH₂CH₂CH₂OCH(CH₃)₂), methylene-t-butoxy (—CH₂—O—C(CH₃)₃) and the like.

The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein. Preferred alkylthioalkoxy groups are alkylene-S-alkyl and include, by way of example, methylenethiomethoxy (—CH₂SCH₃), ethylenethiomethoxy (—CH₂CH₂SCH₃), n-propylene-iso-thiopropoxy (—CH₂CH₂CH₂SCH(CH₃)₂), methylene-t-thiobutoxy (—CH₂SC(CH₃)₃) and the like.

The term “alkenyl” refers to a monoradical of a branched or unbranched unsaturated hydrocarbon group preferably having from 2 to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of vinyl unsaturation. Preferred alkenyl groups include ethenyl (—CH═CH₂), n-propenyl (—CH₂CH═CH₂), iso-propenyl (—C(CH₃)═CH₂), and the like.

The term “substituted alkenyl” refers to an alkenyl group as defined above having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “alkenylene” refers to a diradical of a branched or unbranched unsaturated hydrocarbon group preferably having from 2 to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of vinyl unsaturation. This term is exemplified by groups such as ethenylene (—CH═CH—), the propenylene isomers (e.g., —CH₂CH═CH— and —C(CH₃)═CH—) and the like.

The term “substituted alkenylene” refers to an alkenylene group as defined above having from 1 to 5 substituents, and preferably from 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Additionally, such substituted alkenylene groups include those where 2 substituents on the alkenylene group are fused to form one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fused to the alkenylene group.

The term “alkynyl” refers to a monoradical of an unsaturated hydrocarbon preferably having from 2 to 40 carbon atoms, more preferably 2 to 20 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of acetylene (triple bond) unsaturation. Preferred alkynyl groups include ethynyl (—C≡CH), propargyl (—CH₂C≡CH) and the like.

The term “substituted alkynyl” refers to an alkynyl group as defined above having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “alkynylene” refers to a diradical of an unsaturated hydrocarbon preferably having from 2 to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of acetylene (triple bond) unsaturation. Preferred alkynylene groups include ethynylene (—C≡C—), propargylene (—CH₂C≡C—) and the like.

The term “substituted alkynylene” refers to an alkynylene group as defined above having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “acyl” refers to the groups HC(O)—, alkyl-C(O)—, substituted alkyl-C(O)—, cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—, cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—, aryl-C(O)—, heteroaryl-C(O)— and heterocyclic-C(O)— where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “acylamino” or “aminocarbonyl” refers to the group —C(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, heterocyclic or where both R groups are joined to form a heterocyclic group (e.g., morpholino) wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “aminoacyl” refers to the group —NRC(O)R where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “aminoacyloxy” or “alkoxycarbonylamino” refers to the group —NRC(O)OR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substituted alkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—, aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclic-C(O)O— wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.

The term “aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to 20 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryl). Preferred aryls include phenyl, naphthyl and the like.

Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents, preferably 1 to 3 substituents, selected from the group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl. Preferred aryl substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.

The term “aryloxy” refers to the group aryl-O— wherein the aryl group is as defined above including optionally substituted aryl groups as also defined above.

The term “arylene” refers to the diradical derived from aryl (including substituted aryl) as defined above and is exemplified by 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, 1,2-naphthylene and the like.

The term “amino” refers to the group —NH₂.

The term “substituted amino refers to the group —NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic provided that both R's are not hydrogen.

The term “carboxyalkyl” or “alkoxycarbonyl” refers to the groups “—C(O)O-alkyl”, “—C(O)O-substituted alkyl”, “—C(O)O-cycloalkyl”, “—C(O)O-substituted cycloalkyl”, “—C(O)O-alkenyl”, “—C(O)O-substituted alkenyl”, “—C(O)O-alkynyl” and “—C(O)O-substituted alkynyl” where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl and substituted alkynyl alkynyl are as defined herein.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “cycloalkenyl” refers to cyclic alkenyl groups of from 4 to 20 carbon atoms having a single cyclic ring and at least one point of internal unsaturation. Examples of suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

The term “heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring).

Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, preferably 1 to 3 substituents, selected from the group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl. Preferred aryl substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl). Preferred heteroaryls include pyridyl, pyrrolyl and furyl.

The term “heteroaryloxy” refers to the group heteroaryl-O—.

The term “heteroarylene” refers to the diradical group derived from heteroaryl (including substituted heteroaryl), as defined above, and is exemplified by the groups 2,6-pyridylene, 2,4-pyridiylene, 1,2-quinolinylene, 1,8-quinolinylene, 1,4-benzofuranylene, 2,5-pyridnylene, 2,5-indolenyl and the like.

The term “heterocycle” or “heterocyclic” refers to a monoradical saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to 40 carbon atoms and from 1 to 10 hetero atoms, preferably 1 to 4 heteroatoms, selected from nitrogen, sulfur, phosphorus, and/or oxygen within the ring.

Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Such heterocyclic groups can have a single ring or multiple condensed rings. Preferred heterocyclics include morpholino, piperidinyl, and the like.

Examples of nitrogen heterocycles and heteroaryls include, but are not limited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, morpholino, piperidinyl, tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen containing heterocycles.

The term “heterocyclooxy” refers to the group heterocyclic-O—.

The term “thioheterocyclooxy” refers to the group heterocyclic-S—.

The term “heterocyclene” refers to the diradical group formed from a heterocycle, as defined herein, and is exemplified by the groups 2,6-morpholino, 2,5-morpholino and the like.

The term “oxyacylamino” or “aminocarbonyloxy” refers to the group —OC(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “spiro-attached cycloalkyl group” refers to a cycloalkyl group attached to another ring via one carbon atom common to both rings.

The term “thiol” refers to the group —SH.

The term “thioalkoxy” refers to the group —S-alkyl.

The term “substituted thioalkoxy” refers to the group —S-substituted alkyl.

The term “thioaryloxy” refers to the group aryl-S— wherein the aryl group is as defined above including optionally substituted aryl groups also defined above.

The term “thioheteroaryloxy” refers to the group heteroaryl-S— wherein the heteroaryl group is as defined above including optionally substituted aryl groups as also defined above.

As to any of the above groups which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the compounds of this invention include all stereochemical isomers arising from the substitution of these compounds, whether the isomers are those arising in the ligands, the linkers, or the multivalent constructs including the ligands and linkers.

The term “pharmaceutically-acceptable salt” refers to salts which retain the biological effectiveness and properties of the multibinding compounds of this invention and which are not biologically or otherwise undesirable. In many cases, the multibinding compounds of this invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically-acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases, include by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, such as alkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines, di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenyl amines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines, di(substituted alkenyl) amines, tri(substituted alkenyl) amines, cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines, substituted cycloalkyl amines, disubstituted cycloalkyl amine, trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl) amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines, disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines, aryl amines, diaryl amines, triaryl amines, heteroaryl amines, diheteroaryl amines, triheteroaryl amines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amines where at least two of the substituents on the amine are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic, and the like. Also included are amines where the two or three substituents, together with the amino nitrogen, form a heterocyclic or heteroaryl group.

Examples of suitable amines include, by way of example only, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl) amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like. It should also be understood that other carboxylic acid derivatives would be useful in the practice of this invention, for example, carboxylic acid amides, including carboxamides, lower alkyl carboxamides, dialkyl carboxamides, and the like.

Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like.

The term “pharmaceutically-acceptable cation” refers to the cation of a pharmaceutically-acceptable salt.

The term “protecting group” or “blocking group” refers to any group which when bound to one or more hydroxyl, thiol, amino or carboxyl groups of the compounds (including intermediates thereof) prevents reactions from occurring at these groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl, thiol, amino or carboxyl group. The particular removable blocking group employed is not critical and preferred removable hydroxyl blocking groups include conventional substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, t-butyl-diphenylsilyl and any other group that can be introduced chemically onto a hydroxyl functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product.

Preferred removable thiol blocking groups include disulfide groups, acyl groups, benzyl groups, and the like.

Preferred removable amino blocking groups include conventional substituents such as t-butoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ), fluorenylmethoxycarbonyl (FMOC), allyloxycarbonyl (ALOC), and the like which can be removed by conventional conditions compatible with the nature of the product.

Preferred carboxyl protecting groups include esters such as methyl, ethyl, propyl, t-butyl etc. which can be removed by mild conditions compatible with the nature of the product.

The term “optional” or “optionally” means that the subsequently described event, circumstance or substituent may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The term “ligand” as used herein denotes a compound that is a polymyxin, circulin or octapeptin antibiotic or other suitable compound which exhibits multibinding properties toward LPS present in certain bacteria, especially Gram-negative bacteria; and/or which demonstrates antibacterial properties, or a synthon thereof. The specific region or regions of the ligand which binds to the LPS present in certain bacteria, especially Gram-negative bacteria is designated as the “ligand domain”. A ligand may be either capable of binding to LPS by itself, or may require the presence of one or more non-ligand components for binding (e.g., Ca⁺², Mg⁺² or a water molecule is required for the binding of a ligand to various ligand binding sites).

The polymyxins are a family of antibiotics that are particularly active against Gram-negative bacteria. The polymyxins are typified by polymyxins B1, B2, and polymyxin E, which is also known as colistin (FIG. 1). Polymyxins are described, for example, in D. A. Storm, K. S. Rosenthal, and P. E. Swanson, Ann. Rev. Biochem. 1977, 46, 723-763, the contents of which are hereby incorporated by reference.

Examples of ligands useful in this invention are described herein. Those skilled in the art will appreciate that portions of the ligand structure that are not essential for specific molecular recognition and binding activity may be varied substantially, replaced or substituted with unrelated structures (for example, with ancillary groups as defined below) and, in some cases, omitted entirely without affecting the binding interaction. The primary requirement for a ligand is that it has a ligand domain as defined above. It is understood that the term ligand is not intended to be limited to compounds known to be useful as antibiotics (e.g., known drugs). Those skilled in the art will understand that the term ligand can equally apply to a molecule that is not normally associated with the ability to bind to LPS. In addition, it should be noted that ligands that exhibit marginal activity or lack useful activity as monomers can be highly active as multivalent compounds because of the benefits conferred by multivalency.

The term “multibinding compound or agent” refers to a compound that is capable of multivalency, as defined below, and which has 2-10 ligands covalently bound to one or more linkers which may be the same or different. Multibinding compounds provide a biological and/or therapeutic effect greater than the aggregate of unlinked ligands equivalent thereto which are made available for binding. That is to say that the biological and/or therapeutic effect of the ligands attached to the multibinding compound is greater than that achieved by the same amount of unlinked ligands made available for binding to the ligand binding sites. The phrase “increased biological or therapeutic effect” includes, for example: increased affinity, increased selectivity for target, increased specificity for target, increased potency, increased efficacy, decreased toxicity, improved duration of activity or action, decreased side effects, increased therapeutic index, improved bioavailibity, improved pharmacokinetics, improved activity spectrum, and the like. The multibinding compounds of this invention will exhibit at least one and preferably more than one of the above-mentioned effects.

The term “potency” refers to the minimum concentration at which a ligand is able to achieve a desirable biological or therapeutic effect. The potency of a ligand is typically proportional to its affinity for its ligand binding site. In some cases, the potency may be non-linearly correlated with its affinity. In comparing the potency of two drugs, e.g., a multibinding agent and the aggregate of its unlinked ligand, the dose-response curve of each is determined under identical test conditions (e.g., in an in vitro or in vivo assay, in an appropriate animal model). The finding that the multibinding agent produces an equivalent biological or therapeutic effect at a lower concentration than the aggregate unlinked ligand is indicative of enhanced potency.

The term “univalency” as used herein refers to a single binding interaction between one ligand as defined herein with one ligand binding site as defined herein. It should be noted that a compound having multiple copies of a ligand (or ligands) exhibit univalency when only one ligand is interacting with a ligand binding site. Examples of univalent interactions are depicted below.

The term “multivalency” as used herein refers to the concurrent binding of from 2 to 10 linked ligands (which may be the same or different) and two or more corresponding ligand binding sites on one or more LPS molecules in Gram-positive bacteria, which may be the same or different.

For example, two ligands connected through a linker that bind concurrently to two ligand binding sites would be considered as bivalency; three ligands thus connected would be an example of trivalency. An example of trivalent binding, illustrating a multibinding compound bearing three ligands versus a monovalent binding interaction, is shown below:

It should be understood that all compounds that contain multiple copies of a ligand attached to a linker or to linkers do not necessarily exhibit the phenomena of multivalency, i.e., that the biological and/or therapeutic effect of the multibinding agent is greater than the sum of the aggregate of unlinked ligands made available for binding to the ligand binding site. For multivalency to occur, the ligands that are connected by a linker or linkers have to be presented to their ligand binding sites by the linker(s) in a specific manner in order to bring about the desired ligand-orienting result, and thus produce a multibinding event.

The term “selectivity” or “specificity” is a measure of the binding preferences of a ligand for different ligand binding sites. The selectivity of a ligand with respect to its target ligand binding site relative to another ligand binding site is given by the ratio of the respective values of K_(d) (i.e., the dissociation constants for each ligand-LPS complex) or, in cases where a biological effect is observed below the K_(d), the ratio of the respective EC₅₀'s (i.e., the concentrations that produce 50% of the maximum response for the ligand interacting with the two distinct ligand binding sites).

The term “ligand binding site” denotes the site on the bacteria that recognizes a ligand domain and provides a binding partner for the ligand, namely the LPS present in certain bacteria, especially Gram-negative bacteria. The ligand binding site may be defined by monomeric or multimeric structures. This interaction may be capable of producing a unique biological effect, for example, modulatory effects, antibacterial effects, may maintain an ongoing biological event, and the like.

It should be recognized that the ligand binding sites of the LPS that participate in biological multivalent binding interactions are constrained to varying degrees by their intra- and inter-molecular associations (e.g., such macromolecular structures may be covalently joined to a single structure, noncovalently associated in a multimeric structure, embedded in a membrane or polymeric matrix, and so on) and therefore have less translational and rotational freedom than if the same structures were present as monomers in solution.

The term “inert organic solvent” means a solvent which is inert under the conditions of the reaction being described in conjunction therewith including, by way of example only, benzene, toluene, acetonitrile, tetrahydrofuran, dimethylformamide, chloroform, methylene chloride, diethyl ether, ethyl acetate, acetone, methylethyl ketone, methanol, ethanol, propanol, isopropanol, t-butanol, dioxane, pyridine, and the like. Unless specified to the contrary, the solvents used in the reactions described herein are inert solvents.

The term “treatment” refers to any treatment of a pathologic condition in a mammal, particularly a human, and includes:

(i) preventing the pathologic condition from occurring in a subject which may be predisposed to the condition but has not yet been diagnosed with the condition and, accordingly, the treatment constitutes prophylactic treatment for the disease condition;

(ii) inhibiting the pathologic condition, i.e., arresting its development;

(iii) relieving the pathologic condition, i.e., causing regression of the pathologic condition; or

(iv) relieving the conditions mediated by the pathologic condition.

The term “pathologic condition which is modulated by treatment with a ligand” covers all disease states (i.e., pathologic conditions) which are generally acknowledged in the art to be usefully treated with polymyxin, circulin or octapeptin antibiotics in general, and those disease states which have been found to be usefully treated by a specific multibinding compound of our invention. Such disease states include, by way of example only, Gram-negative bacterial infections.

Examples of bacteria which can be treated with the compositions and methods described herein include gram-negative rods such as enteric gram-negative rods, Pseudomonas, curved gram-negative rods, parvobacteria and Haemophilus, gram-negative cocci such as Neisseria, obligate anaerobes such as Clostridium, non-sporing anaerobes, and unusual bacteria such as spirochaetes, rickettsia and chlamydia. Specific examples of bacterial diseases include chlamydia, gonorrhea, salmonellosis, shigellosis, tuberculosis, syphilis, bacterial pneumonia, bacterial sepsis, urinary tract infections, bacterial upper respiratory tract infections, otitis media, and lyme disease.

Bacteria may be designated as gram-positive and gram-negative, based on the staining of their cell walls with Gram's stain. In gram-positive bacteria, proteins are secreted into the surrounding medium, whereas in gram-negative bacteria, secretion occurs into the periplasmic space between the cytoplasmic and outer membranes (Model and Russel (1990) Cell 61:739-741; Schatz and Beckwith (1990) Ann. Rev. Genet. 24:215-248).

The term “therapeutically effective amount” refers to that amount of multibinding compound which is sufficient to effect treatment, as defined above, when administered to a mammal in need of such treatment. The therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

The term “linker”, identified where appropriate by the symbol X, X′ or X″, refers to a group or groups that covalently links from 2 to 10 ligands (as identified above) in a manner that provides for a compound capable of multivalency. Among other features, the linker is a ligand-orienting entity that permits attachment of multiple copies of a ligand (which may be the same or different) thereto. In some cases, the linker may itself be biologically active. The term “linker” does not, however, extend to cover solid inert supports such as beads, glass particles, fibers, and the like. But it is understood that the multibinding compounds of this invention can be attached to a solid support if desired. For example, such attachment to solid supports can be made for use in separation and purification processes and similar applications.

The term “library” refers to at least 3, preferably from 10² to 10⁹ and more preferably from 10² to 10⁴ multimeric compounds. Preferably, these compounds are prepared as a multiplicity of compounds in a single solution or reaction mixture which permits facile synthesis thereof. In one embodiment, the library of multimeric compounds can be directly assayed for multibinding properties. In another embodiment, each member of the library of multimeric compounds is first isolated and, optionally, characterized. This member is then assayed for multibinding properties.

The term “collection” refers to a set of multimeric compounds which are prepared either sequentially or concurrently (e.g., combinatorially). The collection comprises at least 2 members; preferably from 2 to 10⁹ members and still more preferably from 10 to 10⁴ members.

The term “multimeric compound” refers to compounds comprising from 2 to 10 ligands covalently connected through at least one linker which compounds may or may not possess multibinding properties (as defined herein).

The term “pseudohalide” refers to functional groups which react in displacement reactions in a manner similar to a halogen. Such functional groups include, by way of example, mesyl, tosyl, azido and cyano groups.

The extent to which multivalent binding is realized depends upon the efficiency with which the linker or linkers that joins the ligands presents these ligands to the array of available ligand binding sites. Beyond presenting these ligands for multivalent interactions with ligand binding sites, the linker or linkers spatially constrains these interactions to occur within dimensions defined by the linker or linkers. Thus, the structural features of the linker (valency, geometry, orientation, size, flexibility, chemical composition, etc.) are features of multibinding agents that play an important role in determining their activities.

The linkers used in this invention are selected to allow multivalent binding of ligands to the ligand binding sites as defined herein, namely, the LPS present in certain bacteria, especially Gram-negative bacteria.

The ligands are covalently attached to the linker or linkers using conventional chemical techniques providing for covalent linkage of the ligand to the linker or linkers. Reaction chemistries resulting in such linkages are well known in the art and involve the use of complementary functional groups on the linker and ligand. Preferably, the complementary functional groups on the linker are selected relative to the functional groups available on the ligand for bonding or which can be introduced onto the ligand for bonding. Again, such complementary functional groups are well known in the art. For example, reaction between a carboxylic acid of either the linker or the ligand and a primary or secondary amine of the ligand or the linker in the presence of suitable, well-known activating agents results in formation of an amide bond covalently linking the ligand to the linker; reaction between an amine group of either the linker or the ligand and a sulfonyl halide of the ligand or the linker results in formation of a sulfonamide bond covalently linking the ligand to the linker; and reaction between an alcohol or phenol group of either the linker or the ligand and an alkyl or aryl halide of the ligand or the linker results in formation of an ether bond covalently linking the ligand to the linker.

Table I below illustrates numerous complementary reactive groups and the resulting bonds formed by reaction there between.

TABLE I Representative Complementary Binding Chemistries First Reactive Group Second Reactive Group Linkage hydroxyl isocyanate urethane amine epoxide β-hydroxyamine sulfonyl halide amine sulfonamide carboxyl amine amide hydroxyl alkyl/aryl halide ether aldehyde amine/NaCNBH₄ amine ketone amine/NaCNBH₄ amine amine isocyanate urea

The linker is attached to the ligand at a position that retains ligand domain-ligand binding site interaction and specifically which permits the ligand domain of the ligand to orient itself to bind to the ligand binding site. Such positions and synthetic protocols for linkage are well known in the art. The term linker embraces everything that is not considered to be part of the ligand.

The relative orientation in which the ligand domains are displayed derives from the particular point or points of attachment of the ligands to the linker, and on the framework geometry. The determination of where acceptable substitutions can be made on a ligand is typically based on prior knowledge of structure-activity relationships (SAR) of the ligand and/or congeners and/or structural information about ligand-LPS complexes (e.g., X-ray crystallography, NMR, and the like). Such positions and the synthetic methods for covalent attachment are well known in the art. Following attachment to the selected linker (or attachment to a significant portion of the linker, for example 2-10 atoms of the linker), the univalent linker-ligand conjugate may be tested for retention of activity in the relevant assay.

Suitable linkers and ligands are discussed more fully below.

At present, it is preferred that the multibinding agent is a bivalent compound, e.g., two ligands which are covalently linked to linker X.

Methodology

The linker, when covalently attached to multiple copies of the ligands, provides a biocompatible, substantially non-immunogenic multibinding compound. The biological activity of the multibinding compound is highly sensitive to the valency, geometry, composition, size, flexibility or rigidity, etc. of the linker and, in turn, on the overall structure of the multibinding compound, as well as the presence or absence of anionic or cationic charge, the relative hydrophobicity/hydrophilicity of the linker, and the like on the linker. Accordingly, the linker is preferably chosen to maximize the biological activity of the multibinding compound. The linker may be chosen to enhance the biological activity of the molecule. In general, the linker may be chosen from any organic molecule construct that orients two or more ligands to their ligand binding sites to permit multivalency. In this regard, the linker can be considered as a “framework” on which the ligands are arranged in order to bring about the desired ligand-orienting result, and thus produce a multibinding compound.

For example, different orientations can be achieved by including in the framework groups containing mono- or polycyclic groups, including aryl and/or heteroaryl groups, or structures incorporating one or more carbon-carbon multiple bonds (alkenyl, alkenylene, alkynyl or alkynylene groups). Other groups can also include oligomers and polymers which are branched- or straight-chain species. In preferred embodiments, rigidity is imparted by the presence of cyclic groups (e.g., aryl, heteroaryl, cycloalkyl, heterocyclic, etc.). In other preferred embodiments, the ring is a six or ten member ring. In still further preferred embodiments, the ring is an aromatic ring such as, for example, phenyl or naphthyl.

Different hydrophobic/hydrophilic characteristics of the linker as well as the presence or absence of charged moieties can readily be controlled by the skilled artisan. For example, the hydrophobic nature of a linker derived from hexamethylene diamine (H₂N(CH₂)₆NH₂) or related polyamines can be modified to be substantially more hydrophilic by replacing the alkylene group with a poly(oxyalkylene) group such as found in the commercially available “Jeffamines”. By controlling the hydrophilicity/hydrophobicity, the ability of the compounds to cross the blood/brain barrier can be controlled. This can be important when one wishes to maximize or minimize CNS effects.

Examples of molecular structures in which the above bonding patterns could be employed as components of the linker are shown below.

The identification of an appropriate framework geometry and size for ligand domain presentation are important steps in the construction of a multibinding compound with enhanced activity. Systematic spatial searching strategies can be used to aid in the identification of preferred frameworks through an iterative process. Numerous strategies are known to those skilled in the art of molecular design and can be used for preparing compounds of this invention.

It is to be noted that “core” structures other than those shown here can be used for determining the optimal framework display orientation of the ligands. The process may require the use of multiple copies of the same central “core” structure or combinations of different types of display cores.

The above-described process can be extended to trimers and compounds of higher valency.

Assays of each of the individual compounds of a collection generated as described above will lead to a subset of compounds with the desired enhanced activities (e.g., potency, selectivity, etc.). The analysis of this subset using a technique such as Ensemble Molecular Dynamics will provide a framework orientation that favors the properties desired. A wide diversity of linkers is commercially available (see, e.g., Available Chemical Directory (ACD)). Many of the linkers that are suitable for use in this invention fall into this category. Other can be readily synthesized by methods well known in the art and/or are described below.

Having selected a preferred framework geometry, the physical properties of the linker can be optimized by varying the chemical composition thereof. The composition of the linker can be varied in numerous ways to achieve the desired physical properties for the multibinding compound.

It can therefore be seen that there is a plethora of possibilities for the composition of a linker. Examples of linkers include aliphatic moieties, aromatic moieties, steroidal moieties, peptides, and the like. Specific examples are peptides or polyamides, hydrocarbons, aromatic groups, ethers, lipids, cationic or anionic groups, or a combination thereof.

Examples are given below, but it should be understood that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. For example, properties of the linker can be modified by the addition or insertion of ancillary groups into or onto the linker, for example, to change the solubility of the multibinding compound (in water, fats, lipids, biological fluids, etc.), hydrophobicity, hydrophilicity, linker flexibility, antigenicity, stability, and the like. For example, the introduction of one or more poly(ethylene glycol) (PEG) groups onto or into the linker enhances the hydrophilicity and water solubility of the multibinding compound, increases both molecular weight and molecular size and, depending on the nature of the unPEGylated linker, may increase the in vivo retention time. Further PEG may decrease antigenicity and potentially enhances the overall rigidity of the linker.

Ancillary groups which enhance the water solubility/hydrophilicity of the linker and, accordingly, the resulting multibinding compounds are useful in practicing this invention. Thus, it is within the scope of the present invention to use ancillary groups such as, for example, small repeating units of ethylene glycols, alcohols, polyols (e.g., glycerin, glycerol propoxylate, saccharides, including mono-, oligosaccharides, etc.), carboxylates (e.g., small repeating units of glutamic acid, acrylic acid, etc.), amines (e.g., tetraethylenepentamine), and the like) to enhance the water solubility and/or hydrophilicity of the multibinding compounds of this invention. In preferred embodiments, the ancillary group used to improve water solubility/hydrophilicity will be a polyether.

The incorporation of lipophilic ancillary groups within the structure of the linker to enhance the lipophilicity and/or hydrophobicity of the multibinding compounds described herein is also within the scope of this invention. Lipophilic groups useful with the linkers of this invention include, by way of example only, aryl and heteroaryl groups which, as above, may be either unsubstituted or substituted with other groups, but are at least substituted with a group which allows their covalent attachment to the linker. Other lipophilic groups useful with the linkers of this invention include fatty acid derivatives which do not form bilayers in aqueous medium until higher concentrations are reached.

Also within the scope of this invention is the use of ancillary groups which result in the multibinding compound being incorporated or anchored into a vesicle or other membranous structure such as a liposome or a micelle. The term “lipid” refers to any fatty acid derivative that is capable of forming a bilayer or a micelle such that a hydrophobic portion of the lipid material orients toward the bilayer while a hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of phosphato, carboxylic, sulfato, amino, sulfhydryl, nitro and other like groups well known in the art. Hydrophobicity could be conferred by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups of up to 20 carbon atoms and such groups substituted by one or more aryl, heteroaryl, cycloalkyl, and/or heterocyclic group(s). Preferred lipids are phosphoglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidyl-ethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoyl-phosphatidylcholine or dilinoleoylphosphatidylcholine could be used. Other compounds lacking phosphorus, such as sphingolipid and glycosphingolipid families are also within the group designated as lipid. Additionally, the amphipathic lipids described above may be mixed with other lipids including triglycerides and sterols.

The flexibility of the linker can be manipulated by the inclusion of ancillary groups which are bulky and/or rigid. The presence of bulky or rigid groups can hinder free rotation about bonds in the linker or bonds between the linker and the ancillary group(s) or bonds between the linker and the functional groups. Rigid groups can include, for example, those groups whose conformational lability is restrained by the presence of rings and/or multiple bonds within the group, for example, aryl, heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups. Other groups which can impart rigidity include polypeptide groups such as oligo- or polyproline chains.

Rigidity may also be imparted by internal hydrogen bonding or by hydrophobic collapse.

Bulky groups can include, for example, large atoms, ions (e.g., iodine, sulfur, metal ions, etc.) or groups containing large atoms, polycyclic groups, including aromatic groups, non-aromatic groups and structures incorporating one or more carbon-carbon multiple bonds (i.e., alkenes and alkynes). Bulky groups can also include oligomers and polymers which are branched- or straight-chain species. Species that are branched are expected to increase the rigidity of the structure more per unit molecular weight gain than are straight-chain species.

In preferred embodiments, rigidity is imparted by the presence of cyclic groups (e.g., aryl, heteroaryl, cycloalkyl, heterocyclic, etc.). In other preferred embodiments, the linker comprises one or more six-membered rings. In still further preferred embodiments, the ring is an aryl group such as, for example, phenyl or naphthyl.

Rigidity can also be imparted electrostatically. Thus, if the ancillary groups are either positively or negatively charged, the similarly charged ancillary groups will force the presenter linker into a configuration affording the maximum distance between each of the like charges. The energetic cost of bringing the like-charged groups closer to each other will tend to hold the linker in a configuration that maintains the separation between the like-charged ancillary groups. Further ancillary groups bearing opposite charges will tend to be attracted to their oppositely charged counterparts and potentially may enter into both inter- and intramolecular ionic bonds. This non-covalent mechanism will tend to hold the linker into a conformation which allows bonding between the oppositely charged groups. The addition of ancillary groups which are charged, or alternatively, bear a latent charge when deprotected, following addition to the linker, include deprotectation of a carboxyl, hydroxyl, thiol or amino group by a change in pH, oxidation, reduction or other mechanisms known to those skilled in the art which result in removal of the protecting group, is within the scope of this invention.

In view of the above, it is apparent that the appropriate selection of a linker group providing suitable orientation, restricted/unrestricted rotation, the desired degree of hydrophobicity/hydrophilicity, etc. is well within the skill of the art. Eliminating or reducing antigenicity of the multibinding compounds described herein is also within the scope of this invention. In certain cases, the antigenicity of a multibinding compound may be eliminated or reduced by use of groups such as, for example, poly(ethylene glycol).

As explained above, the multibinding compounds described herein comprise 2-10 ligands attached to a linker that links the ligands in such a manner that they are presented to the LPS for multivalent interactions with ligand binding sites thereon/therein. The linker spatially constrains these interactions to occur within dimensions defined by the linker. This and other factors increases the biological activity of the multibinding compound as compared to the same number of ligands made available in monobinding form.

The compounds of this invention are preferably represented by the empirical formula (L)_(p)(X)_(q) where L, X, p and q are as defined above. This is intended to include the several ways in which the ligands can be linked together in order to achieve the objective of multivalency, and a more detailed explanation is described below.

As noted previously, the linker may be considered as a framework to which ligands are attached. Thus, it should be recognized that the ligands can be attached at any suitable position on this framework, for example, at the termini of a linear chain or at any intermediate position.

The simplest and most preferred multibinding compound is a bivalent compound which can be represented as L—X—L, where each L is independently a ligand which may be the same or different and each X is independently the linker. Examples of such bivalent compounds are provided in FIG. 8, where each shaded circle represents a ligand. A trivalent compound could also be represented in a linear fashion, i.e., as a sequence of repeated units L—X—L—X—L, in which L is a ligand and is the same or different at each occurrence, as can X. However, a trimer can also be a radial multibinding compound comprising three ligands attached to a central core, and thus represented as (L)₃X, where the linker X could include, for example, an aryl or cycloalkyl group. Illustrations of trivalent and tetravalent compounds of this invention are found in FIGS. 9 and 10 respectively where, again, the shaded circles represent ligands. Tetravalent compounds can be represented in a linear array, e.g.,

L—X—L—X—L—X—L

in a branched array, e.g.,

(a branched construct analogous to the isomers of butane—n-butyl, iso-butyl, sec-butyl, and t-butyl) or in a tetrahedral array, e.g.,

where X and L are as defined herein. Alternatively, it could be represented as an alkyl, aryl or cycloalkyl derivative as above with four (4) ligands attached to the core linker.

The same considerations apply to higher multibinding compounds of this invention containing 5-10 ligands as illustrated in FIG. 11 where, as before, the shaded circles represent ligands. However, for multibinding agents attached to a central linker such as aryl or cycloalkyl, there is a self-evident constraint that there must be sufficient attachment sites on the linker to accommodate the number of ligands present; for example, a benzene ring could not directly accommodate more than 6 ligands, whereas a multi-ring linker (e.g., biphenyl) could accommodate a larger number of ligands.

Certain of the above described compounds may alternatively be represented as cyclic chains of the form:

and variants thereof.

All of the above variations are intended to be within the scope of the invention defined by the formula (L)_(p)(X)_(q).

With the foregoing in mind, a preferred linker may be represented by the following formula:

—X^(a)—Z—(Y^(a)—Z)_(m)—Y^(b)—Z—X^(a)—

in which:

m is an integer of from 0 to 20;

X^(a) at each separate occurrence is selected from the group consisting of —O—, —S—, —NR—, —C(O)—, —C(O)O—, —C(O)NR—, —C(S), —C(S)O—, —C(S)NR— or a covalent bond where R is as defined below;

Z is at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond;

Y^(a) and Y^(b) at each separate occurrence are selected from the group consisting of:

 —S—S— or a covalent bond;

 in which:

n is 0, 1 or 2; and

R, R′ and R″ at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic.

Additionally, the linker moiety can be optionally substituted at any atom therein by one or more alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic group.

In one embodiment of this invention, the linker (i.e., X, X′ or X″) is selected those shown in Table II:

TABLE II Representative Linkers Linker —HN—(CH₂)₂—NH—C(O)—(CH₂)—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₂—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₃—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₄—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₅—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₆—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₇—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₈—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₉—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₁₀—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₁₁—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₁₂—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—Z—C(O)—NH—(CH₂)₂—NH— where Z is 1,2-phenyl —HN—(CH₂)₂—NH—C(O)—Z—C(O)—NH—(CH₂)₂—NH— where Z is 1,3-phenyl —HN—(CH₂)₂—NH—C(O)—Z—C(O)—NH—(CH₂)₂—NH— where Z is 1,4-phenyl —HN—(CH₂)₂—NH—C(O)—Z—O—Z—C(O)—NH—(CH₂)₂—NH— where Z is 1,4-phenyl —HN—(CH₂)₂—NH—C(O)—(CH₂)₂—CH(NH—C(O)—(CH₂)₈—CH₃)—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)—O—(CH₂)—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—Z—C(O)—NH—(CH₂)₂—NH— where Z is 5-(n-octadecyloxy)-1,3-phenyl —HN—(CH₂)₂—NH—C(O)—(CH₂)₂—CH(NH—C(O)—Z)—C(O)—NH—(CH₂)₂—NH— where Z is 4-biphenyl —HN—(CH₂)₂—NH—C(O)—Z—C(O)—NH—(CH₂)₂—NH— where Z is 5-(n-butyloxy)-1,3-phenyl —HN—(CH₂)₂—NH—C(O)—(CH₂)₈-trans-(CH═CH)—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₂—CH(NH—C(O)—(CH₂)₁₂—CH₃)—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₂—CH(NH—C(O)—Z)—C(O)—NH—(CH₂)₂—NH— where Z is 4-(n-octyl)-phenyl —HN—(CH₂)—Z—O—(CH₂)₆—O—Z—(CH₂)—NH— where Z is 1,4-phenyl —HN—(CH₂)₂—NH—C(O)—(CH₂)₂—NH—C(O)—(CH₂)₃—C(O)—NH—(CH₂)₂—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₂—CH(NH—C(O)—Ph)—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)—N+((CH₂)₉—CH₃)(CH₂—C(O)—NH—(CH₂)₂—NH₂)—(CH₂)—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)—N((CH₂)₉—CH₃)—(CH₂)—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₂—NH—C(O)—(CH₂)₂—NH—C(O)—(CH₂)3—C(O)—NH—(CH₂)₂—C(O)—NH—(CH₂)₂—C(O)—NH— (CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—Z—C(O)—NH—(CH₂)₂—NH— where Z is 5-hydroxy-1,3-phenyl

In another embodiment of this invention, the linker (i.e., X, X′ or X″) has the formula:

wherein

each R^(a) is independently selected from the group consisting of a covalent bond, alkylene, substituted alkylene and arylene;

each R^(b) is independently selected from the group consisting of hydrogen, alkyl and substituted alkyl; and

n′ is an integer ranging from 1 to about 20.

In view of the above description of the linker, it is understood that the term “linker” when used in combination with the term “multibinding compound” includes both a covalently contiguous single linker (e.g., L—X—L) and multiple covalently non-contiguous linkers (L—X—L—X—L) within the multibinding compound.

Ligands

The polymyxins are lipopeptides produced through non-ribosomal biosynthesis by Gram-positive soil bacteria of the genus Bacillus. Common features of the polymyxins are a decapeptide lariat structure that includes five or six L-(α,γ-diaminobutyric acid (DAB) residues, a D-amino acid residue at position 9 (phenylalanine or leucine), and N-terminal acylation. The polymyxins differ in the precise nature of the N-acyl group (6-methyloctanoic acid or 6-methylheptanoic acid) and the identity of amino acid residues at positions 3 (DAB or L-Ser), 6 (D-Phe or D-Leu), and 7 (L-Leu or L-Thr).

The polymyxins, circulins and octapeptins are all related in structure and in their mode of action, and the tenets for increasing the biological benefits of polymyxins through multivalency apply equally well to these agents and to heteromultivalomers bearing at least one polymyxin subunit along with at least one subunit from the octapeptins, circulins, or other antibiotics.

Total chemical syntheses of polymyxins B1 and E were achieved by Vogler et al. as part of efforts to elucidate polymyxin structure, and a polymyxin analog in which all DAB residues were replaced by lysine has been prepared.

Any compound which is a polymyxin, circulin or octapeptin antibiotic or other suitable compound which exhibits multibinding properties toward LPS present in certain bacteria, especially Grain-negative bacteria; and/or which demonstrates antibacterial properties; and which can be covalently linked to a linker can be used as a ligand to prepare the compounds described herein. Such ligands are well known to those of skill in the art. Examples include polymyxin A, polymyxin B1, polymyxin B2, polymyxin D1, polymyxin E1, polymyxin E2, circulin A, octapeptin A1, octapeptin A2, octapeptin A3, octapeptin B1, octapeptin B2, octapeptin B3, octapeptin C1.

These compounds have also been derivatized and the derivatives often show the same or enhanced activity. Structure/activity relationships within the polymyxins have been examined primarily through semi-synthetic modification of the natural products. The key lesson to emerge from these studies is that a positively charged cyclic heptapeptide and a fatty acid chain of intermediate length (containing 8-14 carbon atoms) are crucial structural determinants for antibiotic activity. Additional observations include:

1. Mono-, di- and tri-N-acetyl derivatives of polymyxin B retain antibacterial activity, whereas the tetra- and penta-N-acetyl derivatives were inactive.

2. Expansion of the cyclic heptapeptide ring by one amino acid residue substantially decreases activity.

3. Deacylation of polymyxins decreases antibacterial potency in vitro by a factor of 30. However, it has been shown that deacylated polymyxin decapeptides as well as deacylated, truncated forms of polymyxin bearing as little as the cyclic heptapeptide core retain the ability to sensitize Gram-negative bacteria to hydrophobic antibiotics such as rifampicin, erythromycin, fusidic acid, clindamycin, novobiocin, which would normally not be cell-penetrant.

4. Substitution of the N-acyl group of polymyxin E with longer (C14), straight-chain N-acyl groups provided the greatest level of activity against typically resistant Gram-positive organisms while addition of shorter (C10,12) chains provided optimal potency against Gram-negative organisms. A similar trend was observed with octapeptin analogs.

5. Linear polymyxin analogs exhibit substantially reduced direct antibacterial activity and permeability increasing activity.

Examples of suitable derivatives include those in which the 2,4-diaminobutyric acid residues are derivatized, for example, alkylated or acylated. For example, the 1 position of polymyxin B and related antibiotics can be alkylated or acylated to form ethyl, diethyl, acetyl, glycyl, protected lysyl, and arginyl derivatives, and the 9 position can be alkylated or acylated to form ethyl, diethyl, dimethyl, bis(2-ethanoic acid), dichloroacetyl, hydroxyacetyl, 6-methyl-octanoyl, amino octanoyl, protected lysyl, lysyl, tyrosyl, arginyl, nitro-araginyl, methionyl, or cysteinyl derivatives.

The peptide side chain or fatty acids can also be removed, the fatty acids replaced with other fatty acids, and/or the peptide sequences can be changed. Other modifications include the addition of single amino acids to the 2,4-diaminobutyric acid residues via the formation of amide linkages.

The compounds can also be chemically modified by formylation, Schiff base formation, for example, with indole aldehyde and pyridoxal phosphate, dinitrophenylation, deamination, guanidation, carbamylation and by reaction with 5-amino-2,4-dinitrofluorobenzene. Several formyl derivatives (the mono-, di- and tri- formyl derivatives) of polymyxin B are known to be as active as or more active than the parent molecule (Srinivasa and Ramachandran, Ind. J. Biochem. and Biophys., 14:54-58 (1978).

The ligands described above (and the precursors and analogs thereof) are well-known in the art and can be readily prepared using art-recognized starting materials, reagents and reaction conditions.

Preparation of Multibinding Compounds

The multibinding compounds of this invention can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, N.Y., 1991, and references cited therein.

Any compound which is a polymyxin, circulin or octapeptin antibiotic or other suitable compound which exhibits multibinding properties toward LPS present in certain bacteria, especially Gram-negative bacteria; and/or which demonstrates antibacterial properties, or a suitable synthon thereof, can be used as a ligand in this invention. As discussed above, numerous such compounds are known in the art and any of these known compounds or derivatives thereof may be employed as ligands in this invention. Typically, a compound selected for use as a ligand will have at least one functional group, such as an amino, hydroxyl, thiol or carboxyl group and the like, which allows the compound to be readily coupled to the linker. Compounds having such functionality are either known in the art or can be prepared by routine modification of known compounds using conventional reagents and procedures.

The ligands can be covalently attached to the linker through any available position on the ligands, provided that when the ligands are attached to the linker, at least one of the ligands retains its ability to function as an antibiotic. Certain sites of attachment of the linker to the ligand are preferred based on known structure-activity relationships. Preferably, the linker is attached to a site on the ligand where structure-activity studies show that a wide variety of substituents are tolerated without loss of activity.

The known structure/activity relationships summarized in the preceeding section indicate that polymyxin subunits may be incorporated into multivalent constructs via attachment of the subunits to frameworks via sidechain amine functional groups of DAB residues without abrogating the activity of the individual subunits. Especially preferred linkage chemistries include reaction with activated acid groups on the framework to form amide bonds, reaction with framework isocyanate groups to form carbamates, reaction with imidate salts to form amidines, reaction with aldehydes followed by reduction to form alkyl linkages, or reaction with alkyl halides to form alkyl linkages. Less preferred linkage chemistries include conjugate addition of DAB amino groups with framework maleimide residues to form amino-succinamide linkages, and reaction with 2-iminothiolane to form amidine derivatives bearing pendant thiol groups that may be reacted with framework iodoacetamide or maleimide moieties for attachment.

More particularly, framework carboxylic acids are activated with a carbodiimide, phosphoryl, or uronium ion reagent in the presence of a 2-fold molar excess of polymyxin subunits to carboxyl groups. These and the other preferred coupling reactions are carried out in polar organic solvents such as dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) using tertiary amine activators such as diisopropylethyl amine when appropriate; however, they may also be carried out under buffered aqueous conditions. Multivalomer products are preferably isolated in crude form by precipitation from the reaction mixture by dilution into a non-polar solvent such as diethyl ether. Crude products are preferably purified by recrystallization, normal- or reverse-phase chromatography, size-exclusion chromatography, or ion-exchange chromatography.

The dense packing of polymyxin's phospholipid targets in the two-dimensional LPS layer suggests that optimal frameworks will display polymyxin subunits linearly or in a plane and separated by no more than 20 angstroms.

One preferred embodiment is to connect 2-10 subunits to a somewhat flexible linear framework such as an oligo(peptide) or a substituted oligo(ethylene glycol). Given that polymyxin B has 5 and colistin has 4 distinct DAB residues, there are many unique 64-orientational isomers” for polymyxin in linear arrays. For polymyxin B divalent compounds which include a completely symmetrical framework alone, there are 15 possible orientations when a linker is coupled to one of the five DAB subunits in the polymyxin ligand (FIG. 3).

In a second preferred embodiment, 3-10 ligand subunits are radially displayed through attachment to a framework consisting of multiple “arms” that radiate from a central core structure such as a benzene ring. The arms of these structures are sufficiently long to span adjacent polymyxin binding sites, estimated to be in the range of 5-10 angstroms. As in the previously described linear embodiment, polymyxin subunits are preferably attached to radial frameworks via DAB amino groups, and this provides for great potential structural diversity.

Most preferred are linear polymyxin di- and tri-valent multimeric compounds, and radial polymyxin tri-valent multimeric compounds. While the above discussion makes it clear that there is significant structural complexity in these cases, the complexity is sufficiently tractable that structure/activity relationships involving valency, framework size, framework geometry, preferred subunit attachment point, preferred polymyxin subunit, preferred framework flexibility and composition, can be systematically addressed. Information obtained from these studies may indicate that further biological benefits will accrue to higher-order multivalomers, and these are then prepared using the same general framework types and coupling chemistries.

The preparation of various multibinding compounds is shown in FIGS. 3-7. The representative polymyxin compound used in the Figures is shown in FIG. 2. As shown in FIG. 3, one equivalent of a diacid, diacid halide, or other difunctional carboxylic acid or sulfonic acid derivative is reacted with two equivalents of a polymyxin compound to form amide or sulfonamide linkages between an γ-amine group on a DAB subunit on one polymyxin compound and an γ-amine group on a DAB subunit on a second polymyxin compound with the carboxylic or sulfonic acid groups on the linker. Since the reaction is random, all fifteen isomers would be expected to be formed (formulas 1a-1o).

Alternatively, one can protect all but one of the amine groups (as shown in FIG. 4) and react two equivalents of the protected polymyxin derivative with the single free amine group with one equivalent of a diacid, diacid halide, or other suitable activated carboxylic or sulfonic acid group to form a specific dimeric compound which includes two identical ligands (FIG. 5). The protected groups can then be deprotected (FIG. 6).

In another embodiment, a directed synthesis of a mixed dimer can be performed by protecting all but one of the γ-amine groups on a polymyxin compound, reacting one equivalent of the protected polymyxin compound with one equivalent of a diacid, diacid halide, or other suitable activated carboxylic or sulfonic acid group. This intermediate can then be reacted with an γ-amine group on a second polymyxin compound to form a mixed dimer (FIG. 7).

It will be understood by those skilled in the art that the following additional methods may be used to prepare other multibinding compounds of this invention. Ligand precursors, for example, ligands containing a leaving group or a nucleophilic group, can be covalently linked to a linker precursor containing a nucleophilic group or a leaving group, using conventional reagents and conditions.

Other methods are well known to those of skill in the art for coupling molecules such as the ligands described herein with the linker molecules described herein. For example, two equivalents of ligand precursor with a halide, tosylate, or other leaving group, can be readily coupled to a linker precursor containing two nucleophilic groups, for example, amine groups, to form a dimer. The leaving group employed in this reaction may be any conventional leaving group including, by way of example, a halogen such as chloro, bromo or iodo, or a sulfonate group such as tosyl, mesyl and the like. When the nucleophilic group is a phenol, any base which effectively deprotonates the phenolic hydroxyl group may be used, including, by way of illustration, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydride, sodium hydroxide, potassium hydroxide, sodium ethoxide, triethylamine, diisopropylethylamine and the like. Nucleophilic substition reactions are typically conducted in an inert diluent, such as tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, acetone, 2-butanone, 1-methyl-2-pyrrolidinone and the like. After the reaction is complete, the dimer is typically isolated using conventional procedures, such as extraction, filtration, chromatography and the like.

By way of further illustration, dimers with a hydrophilic linker can be formed using a ligand precursor containing nucleophilic groups and a polyoxyethylene containing leaving groups, for example, poly(oxyethylene) dibromide (where the number of oxyethylene units is typically an integer from 1 to about 20). In this reaction, two molar equivalents of the ligand precursor are reacted with one molar equivalent of the poly(oxyethylene) dibromide in the presence of excess potassium carbonate to afford a dimer. This reaction is typically conducted in N,N-dimethylformamide at a temperature ranging from about 25° C. to about 100° C. for about 6 to about 48 hours.

Alternatively, the linker connecting the ligands may be prepared in several steps. Specifically, a ligand precursor can first be coupled to an “adapter”, i.e., a bifunctional group having a leaving group at one end and another functional group at the other end which allows the adapter to be coupled to a intermediate linker group. In some cases, the functional group used to couple to the intermediate linker is temporarily masked with a protecting group (“PG”). Representative examples of adapters include, by way of illustration, tert-butyl bromoacetate, 1-Fmoc-2-bromoethylamine, 1-trityl-2-bromoethanethiol, 4-iodobenzyl bromide, propargyl bromide and the like. After the ligand precursor is coupled to the adapter and the protecting group is removed from the adapter's functional group (if a protecting group is present) to form an intermediate, two molar equivalents of the intermediate are then coupled with an intermediate linker to form a dimer.

Ligand precursors can be coupled with adapters which include both leaving groups and protecting groups to form protected intermediates. The leaving group employed in this reaction may be any conventional leaving group including, by way of example, a halogen such as chloro, bromo or iodo, or a sulfonate group such as tosyl, mesyl and the like. Similarly, any conventional protecting group may be employed including, by way of example, esters such as the methyl, tert-butyl, benzyl (“Bn”) and 9-fluorenylmethyl (“Fm”) esters.

Protected intermediates can then be deprotected using conventional procedures and reagents to afford deprotected intermediates. For example, tert-butyl esters are readily hydrolyzed with 95% trifluoroacetic acid in dichloromethane; methyl esters can be hydrolyzed with lithium hydroxide in tetrahydrofuiran/water; benzyl esters can be removed by hydrogenolysis in the presence of a catalyst, such as palladium on carbon; and 9-fluorenylmethyl esters are readily cleaved using 20% piperidine in DMF. If desired, other well-known protecting groups and deprotecting procedures may be employed in these reactions to form deprotected intermediates.

Similarly, ligand precursors having an adapter with an amine functional group can be prepared. Ligand precursors can be coupled with adapters which include leaving groups and protected amine groups to afford protected intermediates. The leaving group employed in this reaction may be any conventional leaving group. Similarly, any conventional amine protecting group may be employed including, by way of example, trityl, tert-butoxycarbonyl (“Boc”), benzyloxycarbonyl (“CBZ”) and 9-fluorenylmethor-carbonyl (“Fmoc”). After coupling the adapter to the ligand precursor, the resulting protected intermediate is deprotected to afford a ligand precursor including an amine group using conventional procedures and reagents. For example, a trityl group is readily removed using hydrogen chloride in acetone; a Boc group is removed using 95% trifluoroacetic acid in dichloromethane; a CBZ group can be removed by hydrogenolysis in the presence of a catalyst, such as palladium on carbon; and a 9-fluorenylmethoxycarbonyl group is readily cleaved using 20% piperidine in DMF to afford the deblocked amine. Other well-known amine protecting groups and deprotecting procedures may be employed in these reactions to form amine-containing intermediates and related compounds.

Ligand precursors having an adapter, for example, one including a free carboxylic acid group or a free amine group, can be readily coupled to intermediate linkers having complementary functional groups to form multibinding compounds as described herein. For example, when one component includes a carboxylic acid group, and the other includes an amine group, the coupling reaction typically employs a conventional peptide coupling reagent and is conducted under conventional coupling reaction conditions, typically in the presence of a trialkylamine, such as ethyldiisopropylamine. Suitable coupling reagents for use in this reaction include, by way of example, carbodiimides, such as ethyl-3-(3-dimethylamino)propylcarboiimide (EDC), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and the like, and other well-known coupling reagents, such as N,N′-carbonyldiimidazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and the like. Optionally, well-known coupling promoters, such N-hydroxysuccinimide, 1-hydroxybenzotriazole (HOBT), 1-hydroxy-7-azabenzotriazole (HOAT), N,N-dimethylaminopyridine (DMAP) and the like, may be employed in this reaction. Typically, this coupling reaction is conducted at a temperature ranging from about 0° C. to about 60° C. for about 1 to about 72 hours in an inert diluent, such as THF, to afford the dimer.

The multibinding compounds described herein can also be prepared using a wide variety of other synthetic reactions and reagents. For example, ligand precursors having aryliodide, carboxylic acid, amine and boronic acid functional groups can be prepared. Hydroxymethyl pyrrole can be readily coupled under Mitsunobu reaction conditions to various phenols to provide, after deprotection, functionalized intermediates. The Mitsunobu reaction is typically conducted by reacting hydroxymethyl pyrrole and the appropriate phenol using diethyl azodicarboxylate (DEAD) and triphenylphosphine at ambient temperature for about 48 hours. Deprotection, if necessary, using conventional procedures and reagents then affords the functionalized intermediates.

The functionalized intermediates can be employed in the synthesis of multibinding compounds. For example, aryliodide intermediates can be coupled with bis-boronic acid linkers to provide dimers. Typically, this reaction is conducted by contacting two molar equivalents of the aryliodide and one molar equivalent of the bis-boronic acid in the presence of tetrakis(triphenylphosphine)palladium(0), sodium carbonate and water in refluxing toluene.

Aryliodide intermediates can also be coupled with acrylate intermediates or alkyne intermediate to afford dimers. These reactions are typically conducted by contacting two molar equivalents of aryliodide intermediates with one molar equivalent of either acrylates or alkynes in the presence of dichlorobis(triphenylphosphine)palladium (II), copper (I) iodide and diisopropylethylamine in N,N-dimethylformamide to afford the respective dimers.

As will be readily apparent to those of ordinary skill in the art, the synthetic procedures described herein or those known in the art may be readily modified to afford a wide variety of compounds within the scope of this invention.

Combinatorial Libraries

The methods described herein lend themselves to combinatorial approaches for identifying multimeric compounds which possess multibinding properties.

Specifically, factors such as the proper juxtaposition of the individual ligands of a multibinding compound with respect to the relevant array of binding sites on a target or targets is important in optimizing the interaction of the multibinding compound with its target(s) and to maximize the biological advantage through multivalency. One approach is to identify a library of candidate multibinding compounds with properties spanning the multibinding parameters that are relevant for a particular target. These parameters include: (1) the identity of ligand(s), (2) the orientation of ligands, (3) the valency of the construct, (4) linker length, (5) linker geometry, (6) linker physical properties, and (7) linker chemical functional groups.

Libraries of multimeric compounds potentially possessing multibinding properties (i.e., candidate multibinding compounds) and comprising a multiplicity of such variables are prepared and these libraries are then evaluated via conventional assays corresponding to the ligand selected and the multibinding parameters desired. Considerations relevant to each of these variables are set forth below:

Selection of Ligand(s)

A single ligand or set of ligands is (are) selected for incorporation into the libraries of candidate multibinding compounds which library is directed against a particular biological target or targets, i.e., the LPS present in certain bacteria, especially Gram-negative bacteria. The only requirement for the ligands chosen is that they are capable of interacting with the selected target(s). Thus, ligands may be known drugs, modified forms of known drugs, substructures of known drugs or substrates of modified forms of known drugs (which are competent to interact with the target), or other compounds. Ligands are preferably chosen based on known favorable properties that may be projected to be carried over to or amplified in multibinding forms. Favorable properties include demonstrated safety and efficacy in human patients, appropriate PK/ADME profiles, synthetic accessibility, and desirable physical properties such as solubility, logP, etc. However, it is crucial to note that ligands which display an unfavorable property from among the previous list may obtain a more favorable property through the process of multibinding compound formation; i.e., ligands should not necessarily be excluded on such a basis. For example, a ligand that is not sufficiently potent at a particular target so as to be efficacious in a human patient may become highly potent and efficacious when presented in multibinding form. A ligand that is potent and efficacious but not of utility because of a non-mechanism-related toxic side effect may have increased therapeutic index (increased potency relative to toxicity) as a multibinding compound. Compounds that exhibit short in vivo half-lives may have extended half-lives as multibinding compounds. Physical properties of ligands that limit their usefulness (e.g. poor bioavailability due to low solubility, hydrophobicity, hydrophilicity) may be rationally modulated in multibinding forms, providing compounds with physical properties consistent with the desired utility.

Orientation: Selection of Ligand Attachment Points and Linking Chemistry

Several points are chosen on each ligand at which to attach the ligand to the linker. The selected points on the ligand/linker for attachment are functionalized to contain complementary reactive functional groups. This permits probing the effects of presenting the ligands to their target binding site(s) in multiple relative orientations, an important multibinding design parameter. The only requirement for choosing attachment points is that attaching to at least one of these points does not abrogate activity of the ligand. Such points for attachment can be identified by structural information when available. For example, inspection of a co-crystal structure of a ligand bound to its target allows one to identify one or more sites where linker attachment will not preclude the ligand/target interaction. Alternatively, evaluation of ligand/target binding by nuclear magnetic resonance will permit the identification of sites non-essential for ligand/target binding. See, for example, Fesik, et al., U.S. Pat. No. 5,891,643, the disclosure of which is incorporated herein by reference in its entirety. When such structural information is not available, utilization of structure-activity relationships (SAR) for ligands will suggest positions where substantial structural variations are and are not allowed. In the absence of both structural and SAR information, a library is merely selected with multiple points of attachment to allow presentation of the ligand in multiple distinct orientations. Subsequent evaluation of this library will indicate what positions are suitable for attachment.

It is important to emphasize that positions of attachment that do abrogate the activity of the monomeric ligand may also be advantageously included in candidate multibinding compounds in the library provided that such compounds bear at least one ligand attached in a manner which does not abrogate intrinsic activity. This selection derives from, for example, heterobivalent interactions within the context of a single target molecule. For example, consider a ligand bound to its target, and then consider modifying this ligand by attaching to it a second copy of the same ligand with a linker which allows the second ligand to interact with the same target at sites proximal to the first binding site, which include elements of the target that are not part of the formal ligand binding site and/or elements of the matrix surrounding the formal binding site, such as the membrane. Here, the most favorable orientation for interaction of the second ligand molecule may be achieved by attaching it to the linker at a position which abrogates activity of the ligand at the first binding site. Another way to consider this is that the SAR of individual ligands within the context of a multibinding structure is often different from the SAR of those same ligands in momomeric form.

The foregoing discussion focused on bivalent interactions of dimeric compounds bearing two copies of the same ligand joined to a single linker through different attachment points, one of which may abrogate the binding/activity of the monomeric ligand. It should also be understood that bivalent advantage may also be attained with heteromeric constructs bearing two different ligands that bind to common or different targets.

Once the ligand attachment points have been chosen, one identifies the types of chemical linkages that are possible at those points. The most preferred types of chemical linkages are those that are compatible with the overall structure of the ligand (or protected forms of the ligand) readily and generally formed, stable and intrinsically innocuous under typical chemical and physiological conditions, and compatible with a large number of available linkers. Amide bonds, ethers, amines, carbamates, ureas, and sulfonamides are but a few examples of preferred linkages.

Linker Selection

In the library of linkers employed to generate the library of candidate multibinding compounds, the selection of linkers employed in this library of linkers takes into consideration the following factors:

Valency: In most instances the library of linkers is initiated with divalent linkers. The choice of ligands and proper juxtaposition of two ligands relative to their binding sites permits such molecules to exhibit target binding affinities and specificities more than sufficient to confer biological advantage. Furthermore, divalent linkers or constructs are also typically of modest size such that they retain the desirable biodistribution properties of small molecules.

Linker Length: Linkers are chosen in a range of lengths to allow the spanning of a range of inter-ligand distances that encompass the distance preferable for a given divalent interaction. In some instances the preferred distance can be estimated rather precisely from high-resolution structural information of targets. In other instances where high-resolution structural information is not available, one can make use of simple models to estimate the maximum distance between binding sites either on adjacent molecules of LPS or at different locations on the same LPS molecule. In situations where two binding sites are present on the same target (or target subunit for multisubunit targets), preferred linker distances are 2-20 Å, with more preferred linker distances of 3-12 Å. In situations where two binding sites reside on separate target sites, preferred linker distances are 20-100 Å, with more preferred distances of 30-70 Å.

Linker Geometry and Rigidity: The combination of ligand attachment site, linker length, linker geometry, and linker rigidity determine the possible ways in which the ligands of candidate multibinding compounds may be displayed in three dimensions and thereby presented to their binding sites. Linker geometry and rigidity are nominally determined by chemical composition and bonding pattern, which may be controlled and are systematically varied as another spanning function in a multibinding array. For example, linker geometry is varied by attaching two ligands to the ortho, meta, and para positions of a benzene ring, or in cis- or trans-arrangements at the 1,1- vs. 1,2- vs. 1,3- vs. 1,4- positions around a cyclohexane core or in cis- or trans-arrangements at a point of ethylene unsaturation. Linker rigidity is varied by controlling the number and relative energies of different conformational states possible for the linker. For example, a divalent compound bearing two ligands joined by 1,8-octyl linker has many more degrees of freedom, and is therefore less rigid than a compound in which the two ligands are attached to the 4,4′ positions of a biphenyl linker.

Linker Physical Properties: The physical properties of linkers are nominally determined by the chemical constitution and bonding patterns of the linker, and linker physical properties impact the overall physical properties of the candidate multibinding compounds in which they are included. A range of linker compositions is typically selected to provide a range of physical properties (hydrophobicity, hydrophilicity, amphiphilicity, polarization, acidity, and basicity) in the candidate multibinding compounds. The particular choice of linker physical properties is made within the context of the physical properties of the ligands they join and preferably the goal is to generate molecules with favorable PK/ADME properties. For example, linkers can be selected to avoid those that are too hydrophilic or too hydrophobic to be readily absorbed and/or distributed in vivo.

Linker Chemical Functional Groups: Linker chemical functional groups are selected to be compatible with the chemistry chosen to connect linkers to the ligands and to impart the range of physical properties sufficient to span initial examination of this parameter.

Combinatorial Synthesis

Having chosen a set of n ligands (n being determined by the sum of the number of different attachment points for each ligand chosen) and m linkers by the process outlined above, a library of (n!)m candidate divalent multibinding compounds is prepared which spans the relevant multibinding design parameters for a particular target. For example, an array generated from two ligands, one which has two attachment points (A1, A2) and one which has three attachment points (B1, B2, B3) joined in all possible combinations provide for at least 15 possible combinations of multibinding compounds:

A1-A1 A1-A2 A1-B1 A1-B2 A1-B3 A2-A2 A2-B1 A2-B2 A2-B3 B1-B1 B1-B2 B1-B3 B2-B2 B2-B3 B3-B3

When each of these combinations is joined by 10 different linkers, a library of 150 candidate multibinding compounds results.

Given the combinatorial nature of the library, common chemistries are preferably used to join the reactive functionalies on the ligands with complementary reactive functionalities on the linkers. The library therefore lends itself to efficient parallel synthetic methods. The combinatorial library can employ solid phase chemistries well known in the art wherein the ligand and/or linker is attached to a solid support. Alternatively and preferably, the combinatorial libary is prepared in the solution phase. After synthesis, candidate multibinding compounds are optionally purified before assaying for activity by, for example, chromatographic methods (e.g., HPLC).

Analysis of the Library

Various methods are used to characterize the properties and activities of the candidate multibinding compounds in the library to determine which compounds possess multibinding properties. Physical constants such as solubility under various solvent conditions and logD/clogD values can be determined. A combination of NMR spectroscopy and computational methods is used to determine low-energy conformations of the candidate multibinding compounds in fluid media. The ability of the members of the library to bind to the desired target and other targets is determined by various standard methods, which include radioligand displacement assays for receptor and ion channel targets, and kinetic inhibition analysis for many enzyme targets. In vitro efficacy, such as for receptor agonists and antagonists, ion channel blockers, and antimicrobial activity, can also be determined. Pharmacological data, including oral absorption, everted gut penetration, other pharmacokinetic parameters and efficacy data can be determined in appropriate models. In this way, key structure-activity relationships are obtained for multibinding design parameters which are then used to direct future work.

The members of the library which exhibit multibinding properties, as defined herein, can be readily determined by conventional methods. First those members which exhibit multibinding properties are identified by conventional methods as described above including conventional assays (both in vitro and in vivo).

Second, ascertaining the structure of those compounds which exhibit multibinding properties can be accomplished via art recognized procedures. For example, each member of the library can be encrypted or tagged with appropriate information allowing determination of the structure of relevant members at a later time. See, for example, Dower, et al., International Patent Application Publication No. WO 93/06121; Brenner, et al., Proc. Natl. Acad. Sci., USA, 89:5181 (1992); Gallop, et al., U.S. Pat. No. 5,846,839; each of which are incorporated herein by reference in its entirety. Alternatively, the structure of relevant multivalent compounds can also be determined from soluble and untagged libaries of candidate multivalent compounds by methods known in the art such as those described by Hindsgaul, et al., Canadian Patent Application No. 2,240,325 which was published on Jul. 11, 1998. Such methods couple frontal affinity chromatography with mass spectroscopy to determine both the structure and relative binding affinities of candidate multibinding compounds to LPS.

The process set forth above for dimeric candidate multibinding compounds can, of course, be extended to trimeric candidate compounds and higher analogs thereof.

Follow-up Synthesis and Analysis of Additional Libraries

Based on the information obtained through analysis of the initial library, an optional component of the process is to ascertain one or more promising multibinding “lead” compounds as defined by particular relative ligand orientations, linker lengths, linker geometries, etc. Additional libraries can then be generated around these leads to provide for further information regarding structure to activity relationships. These arrays typically bear more focused variations in linker structure in an effort to further optimize target affinity and/or activity at the target (antagonism, partial agonism, etc.), and/or alter physical properties. By iterative redesign/analysis using the novel principles of multibinding design along with classical medicinal chemistry, biochemistry, and pharmacology approaches, one is able to prepare and identify optimal multibinding compounds that exhibit biological advantage towards their targets and as therapeutic agents.

To further elaborate upon this procedure, suitable divalent linkers include, by way of example only, those derived from dicarboxylic acids, disulfonylhalides, dialdehydes, diketones, dihalides, diisocyanates,diamines, diols, mixtures of carboxylic acids, sulfonylhalides, aldehydes, ketones, halides, isocyanates, amines and diols. In each case, the carboxylic acid, sulfonylhalide, aldehyde, ketone, halide, isocyanate, amine and diol functional group is reacted with a complementary functionality on the ligand to form a covalent linkage. Such complementary functionality is well known in the art as illustrated in the following table:

Representative Complementary Binding Chemistries First Reactive Group Second Reactive Group Linkage hydroxyl isocyanate urethane amine epoxide β-hydroxyamine sulfonyl halide amine sulfonamide carboxyl acid amine amide hydroxyl alkyl/aryl halide ether aldehyde amine (+ reducing agent) amine ketone amine (+ reducing agent) amine amine isocyanate urea

The following table illustrates, by way of example, starting materials (identified as X-1 through X-418) that can be used to prepare linkers incorporated in the multibinding compounds of this invention utilizing the chemistry described above. For example, 1,10-decanedicarboxylic acid, X1, can be reacted with 2 equivalents of a ligand carrying an amino group in the presence of a coupling reagent such as DCC to provide a bivalent multibinding compound of formula (I) wherein the ligands are linked via a 1,10-decanediamido linking group.

Exemplary linkers include the following linkers identified as X-1 through X-418 as set forth below:

Pharmaceutical Formulations

Lipopolysaccharides are the primary component of the outer membrane of Gram-negative bacteria; thus, the targets for the compounds described herein exist densely packed on the surface of target organisms. Given this, linkage of 2 or more polymyxin, octapeptin or colistin subunits with an appropriate framework increases both affinity for bacterial outer membranes and specificity for bacterial membranes versus mammalian cell membranes. Furthermore, the compounds described herein are more efficient than the parent compounds at disrupting bacterial membranes, as the multivalent constructs confine multiple membrane disrupting effects within the dimensions, geometries, and other parameters specified by the framework.

The compounds described herein also display enhanced lipid A deactivating ability. Overall, these effects increase the potency and spectrum of action, efficacy in treatment of infections due to Gram-negative organisms, efficacy in preventing or ameliorating inflammatory responses to endotoxin, and duration of action of polymyxin while concurrently reducing toxic side effects. The compounds (and deacylated forms thereof) are also superior at sensitizing Gram-negative organisms to normally inpenetrant hydrophobic antibiotics.

When employed as pharmaceuticals, the compounds described herein are usually administered in the form of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These compounds are effective as both injectable and oral compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.

The administration of these compounds by other than a topical route represents an advance over the prior art. Monovalent polymyxin, circulin or octapeptin antibiotics, when administered systemically, often resulted in nephrotoxicity, and were associated with albuminuria, cellular cylindruria, and azotemia. Other side effects included nausea, vomiting, injection site pain, and skin rashes. The multivalent compounds described herein can be used in lower doses to achieve the same effect, and are not associated with the same side effect profile (i.e., are expected to produce less nephrotoxicity) as the monovalent polymyxin, circulin or octapeptin compositions in the prior art.

This invention also includes pharmaceutical compositions which contain, as the active ingredient, one or more of the compounds described herein associated with pharmaceutically acceptable carriers. In making the compositions of this invention, the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

The compositions are preferably formulated in a unit dosage form, each dosage containing from about 0.001 to about 1 g, more usually about 1 to about 30 mg, of the active ingredient. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Preferably, the compound of formula I above is employed at no more than about 20 weight percent of the pharmaceutical composition, more preferably no more than about 15 weight percent, with the balance being pharmaceutically inert carrier(s).

The active compound is effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It, will be understood, however, that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. Preferably the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

The following formulation examples illustrate representative pharmaceutical compositions of the present invention.

Formulation Example 1

Hard gelatin capsules containing the following ingredients are prepared:

Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch 305.0 Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.

Formulation Example 2

A tablet formula is prepared using the ingredients below:

Quantity Ingredient (mg/tablet) Active Ingredient 25.0 Cellulose, microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing 240 mg.

Formulation Example 3

A dry powder inhaler formulation is prepared containing the following components:

Ingredient Weight % Active Ingredient  5 Lactose 95

The active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.

Formulation Example 4

Tablets, each containing 30 mg of active ingredient, are prepared as follows:

Quantity Ingredient (mg/tablet) Active Ingredient 30.0 mg Starch 45.0 mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone  4.0 mg (as 10% solution in sterile water) Sodium carboxymethyl starch  4.5 mg Magnesium stearate  0.5 mg Talc  1.0 mg Total  120 mg

The active ingredient, starch and cellulose are passed through a No. 20 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders, which are then passed through a 16 mesh U.S. sieve. The granules so produced are dried at 50° to 60° C. and passed through a 16 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 30 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 120 mg.

Formulation Example 5

Capsules, each containing 40 mg of medicament are made as follows:

Quantity Ingredient (mg/capsule) Active Ingredient  40.0 mg Starch 109.0 mg Magnesium stearate  1.0 mg Total 150.0 mg

The active ingredient, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 150 mg quantities.

Formulation Example 6

Suppositories, each containing 25 mg of active ingredient are made as follows:

Ingredient Amount Active Ingredient   25 mg Saturated fatty acid glycerides to 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool.

Formulation Example 7

Suspensions, each containing 50 mg of medicament per 5.0 mL dose are made as follows:

Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose (89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Color q.v. Purified water to 5.0 mL

The active ingredient, sucrose and xanthan gum are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water. The sodium benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume.

Formulation Example 8

A formulation may be prepared as follows:

Quantity Ingredient (mg/capsule) Active Ingredient  15.0 mg Starch 407.0 mg Magnesium stearate  3.0 mg Total 425.0 mg

The active ingredient, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 425.0 mg quantities.

Formulation Example 3

A formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 5.0 mg Corn Oil 1.0 mL

Formulation Example 10

A topical formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 1-10 g Emulsifying Wax 30 g Liquid Paraffin 20 g White Soft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin and emulsifying wax are incorporated and stirred until dissolved. The active ingredient is added and stirring is continued until dispersed. The mixture is then cooled until solid.

Another preferred formulation employed in the methods of the present invention employs transdermal delivery devices (“patches”). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated by reference in its entirety. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

Other suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985).

Utility

The multibinding compounds of this invention are multivalent forms of polymyxin, circulin or octapeptin antibiotics, which (the monovalent forms) are known to mediate bacterial infections. Accordingly, the multibinding compounds and pharmaceutical compositions of this invention are useful in the treatment and prevention of various disorders, in particular, bacterial infections. Examples of bacterial infections which can be treated include, but are not limited to, infections caused by Enterobacter sp., E. coli, Klebsiella sp., Salmonella sp., Pasteurella sp., Bordetella sp., Shigella sp., Pseudomonas aeruginosa, Proteus mirabilis, and Serratia marcesens.

In therapeutic applications, compositions are administered to a patient already suffering from a bacterial infection in an amount sufficient to at least partially reduce the symptoms. Amounts effective for this use will depend on the judgment of the attending clinician depending upon factors such as the degree or severity of the disorder in the patient, the age, weight and general condition of the patient, and the like. The pharmaceutical compositions of this invention may contain more than one compound of the present invention.

As noted above, the compounds administered to a patient are in the form of pharmaceutical compositions described above which can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, etc. These compounds are effective as both injectable and oral deliverable pharmaceutical compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.

The multibinding compounds are more potent, and have fewer side effects than the parent compounds, so less drug needs to be given. Further, many multibinding compounds described herein do not produce the side effects associated with the parent compounds. Accordingly, many of the compounds can be administered systemically without the problems associated with nephrotoxicity and other side effects.

The multibinding compounds of this invention can also be administered in the form of pro-drugs, i.e., as derivatives which are converted into a biologically active compound in vivo. Such pro-drugs will typically include compounds in which, for example, a carboxylic acid group, a hydroxyl group or a thiol group is converted to a biologically liable group, such as an ester, lactone or thioester group which will hydrolyze in vivo to reinstate the respective group.

Combinatorial Chemistry

The compounds can be assayed to identify which of the multimeric ligand compounds possess multibinding properties. First, one identifies a ligand or mixture of ligands which each contain at least one reactive functionality and a library of linkers which each include at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand. Next one prepares a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands with the library of linkers under conditions wherein the complementary functional groups react to form a covalent linkage between the linker and at least two of the ligands. The multimeric ligand compounds produced in the library can be assayed to identify multimeric ligand compounds which possess multibinding properties. The method can also be performed using a library of ligands and a linker or mixture of linkers.

The preparation of the multimeric ligand compound library can be achieved by either the sequential or concurrent combination of the two or more stoichiometric equivalents of the ligands with the linkers. The multimeric ligand compounds can be dimeric, for example, homomeric or heteromeric. A heteromeric ligand compound library can be prepared by sequentially adding a first and second ligand.

Each member of the multimeric ligand compound library can be isolated from the library, for example, by preparative liquid chromatography mass spectrometry (LCMS). The linker or linkers can be flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers of different geometry, acidic linkers, basic linkers, linkers of different polarization and/or polarizability or amphiphilic linkers. The linkers can include linkers of different chain lengths and/or which have different complementary reactive groups. In one embodiment, the linkers are selected to have different linker lengths ranging from about 2 to 100 Å. The ligand or mixture of ligands can have reactive functionality at different sites on the ligands. The reactive functionality can be, for example, carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, pseudohalides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates, and precursors thereof, as long as the reactive functionality on the ligand is complementary to at least one of the reactive groups on the linker so that a covalent linkage can be formed between the linker and the ligand.

A library of multimeric ligand compounds can thus be formed which possesses multivalent properties.

Multimeric ligand compounds possessing multibinding properties can be identified in an iterative method by preparing a first collection or iteration of multimeric compounds by contacting at least two stoichiometric equivalents of the ligand or mixture of ligands which are polymyxin, circulin or octapeptin antibiotics or other suitable compounds which exhibit multibinding properties toward LPS and/or which demonstrate antibacterial properties, with a linker or mixture of linkers, where the ligand or mixture of ligands includes at least one reactive functionality and the linker or mixture of linkers includes at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand. The ligand(s) and linker(s) are reacted under conditions which form a covalent linkage between the linker and at least two of the ligands. The first collection or iteration of multimeric compounds can be assayed to assess which if any of the compounds possess multibinding properties. The process can be repeated until at least one multimeric compound is found to possess multibinding properties. By evaluating the particular molecular constraints which are imparted or are consistent with imparting multibinding properties to the multimeric compound or compounds in the first iteration, a second collection or iteration of multimeric compounds which elaborates upon the particular molecular constraints can be assayed, and the steps optionally repeated to further elaborate upon said molecular constraints. For example, the steps can be repeated from between 2 and 50 times, more preferably, between 5 and 50 times.

The following preparative and biological examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of this invention. Unless otherwise stated, all temperatures are in degrees Celsius.

EXAMPLES

In the examples below, the following abbreviations have the following meanings. If an abbreviation is not defined, it has its generally accepted meaning.

Å = Angstroms cm = centimeter DCC = dicyclohexyl carbodiimide DMF = N,N-dimethylformamide DMSO = dimethylsulfoxide EDTA = ethylenediaminetetraacetic acid g = gram HPLC = high performance liquid chromatography MEM = minimal essential medium mg = milligram MIC = minimum inhibitory concentration min = minute mL = milliliter mm = millimeter mmol = millimol N = normal THF = tetrahydrofuran μL = microliters μm = microns

Biological Examples Example 1

In Vitro Determination of Antibacterial Activity

Bacterial strains are obtained from either the American Type Tissue Culture Collection (ATCC), Stanford University Hospital (SU), Kaiser Permanente Regional Laboratory in Berkeley (KPB), Massachusetts General Hospital (MGH), the Centers for Disease Control (CDC), the San Francisco Veterans'Administration Hospital (SFVA) or the University of California San Francisco Hospital (UCSF). Vancomycin resistant enterococci are phenotyped as Van A or Van B based on their sensitivity to teicoplanin. Some vancomycin resistant enterococci that had been genotyped as Van A, Van B, Van C1 or Van C2 are obtained from the Mayo Clinic.

Minimal inhibitory concentrations (MICs) are measured in a microdilution broth procedure under NCCLS guidelines. Routinely the compositions are serially diluted into Mueller-Hinton broth in 96-well microtiter plates. Overnight cultures of bacterial strains are diluted based on absorbance at 600 nm so that the final concentration in each well is 5×10⁵ cfu/ml. Plates are returned to a 35° C. incubator. The following day (or 24 hours in the case of Enterococci strains), MICs are determined by visual inspection of the plates. Strains routinely tested in the initial screen include gram-negative rods such as enteric gram-negative rods, Pseudomonas, curved gram-negative rods, and Haemophilus, gram-negative cocci such as Neisseria. Because of the inability of PSSP and PSRP to grow well in Mueller-Hinton broth, MICs with those strains are determined using either TSA broth supplemented with defibrinated blood or blood agar plates. Compositions which had significant activity against the strains mentioned above are then tested for MIC values in a larger panel of clinical isolates including those of Escherichia coli and Pseudomonas aeruginosa.

Determination of Kill Time

Experiments to determine the time required to kill the bacteria are conducted as described in Lorian. These experiments are conducted normally with both Escherichia coli and Pseudomonas aeruginosa strains. Briefly, several colonies are selected from an agar plate and grown at 35° C. under constant agitation until it achieved a turbidity of approximately 1.5 and 10⁸ CFU/ml. The sample is then diluted to about 6×106 CFU/ml and incubation at 35° C. under constant agitation is continued. At various times aliquots are removed and five ten-fold serial dilutions are performed. The spread plate method is used to determine the number of colony forming units (CFUs).

Example 2

In Vivo Determination of Antibacterial Activity

Acute tolerability studies in mice.

In these studies, compositions including the active compound are administered either intravenously or subcutaneously and observed for 5-15 minutes. If there are no adverse effects, the dose is increased in a second group of mice. This dose incrementation is continued until mortality occurs, or the dose is maximized. Generally, dosing begins at 20 mg/kg and is increased by 20 mg/kg each time until the maximum tolerated dose (MTD) is achieved.

Bioavailability studies in mice

Mice are administered the composition either intravenously or subcutaneously at a therapeutic dose (in general, approximately 50 mg/kg). Groups of animals are placed in metabolic cages so that urine and feces could be collected for analysis. Groups of animals (n=3) are sacrificed at various times (10 min, 1 hour and 4 hours). Blood is collected by cardiac puncture and the following organs are harvested—lung, liver, heart, brain, kidney, and spleen. Tissues are weighed and prepared for HPLC analysis. HPLC analysis on the tissue homogenates and fluids is used to determine the concentration of the active compound present. Metabolic products resulting from changes to the active compound are also determined at this juncture.

Mouse septecemia model.

In this model, an appropriately virulent strain of bacteria (most commonly Escherichia coli or Pseudomonas aeruginosa) is administered to mice (N=5 to 10 mice per group) intraperitoneally. The bacteria is combined with hog gastric mucin to enhance virulence. The dose of bacteria (normally 10⁵-10⁷) is that sufficient to induce mortality in all of the mice over a three day period. One hour after the bacteria is administered, the active compound is administered in a single dose either IV or subcutaneously. Each dose is administered to groups of 5 to 10 mice, at doses that typically ranged from a maximum of about 20 mg/kg to a minimum of less than 1 mg/kg. A positive control (normally vancomycin with vancomycin sensitive strains) is administered in each experiment. The dose at which approximately 50% of the animals are saved is calculated from the results.

Pharmacokinetic studies

The rate at which the active compound is removed from the blood can be determined in either rats or mice. In rats, the test animals are cannulated in the jugular vein. The composition including the active compound is administered via tail vein injection, and at various time points (normally 5, 15, 30,60 minutes and 2,4,6 and 24 hours) blood is withdrawn from the cannula. In mice, the composition is also administered via tail vein injection, and at various time points. Blood is normally obtained by cardiac puncture. The concentration of the remaining composition is determined by HPLC.

Preparative Examples Example 1

Preparation of (3) Through (17), Compounds of Formula I-a Through I-o Via Scheme A

Azelaic acid (2) (20 mmol) is dissolved in DMF and treated sequentially with hydroxybenzotriazole (50 mmol), diisopropylethyl amine (40 mmol) and PyBOP (40 mmol). After stirring for 15 minutes at room temperature, the activated diacid is treated with compound (1) (40 mmol) and the coupling reaction is stirred overnight at room temperature. Volatiles are removed under vacuum and the crude is fractionated by reverse-phase HPLC to afford the title products after lyopholization of the appropriate fractions. The desired material is then purified from this mixture using HPLC to afford the title products (3) through (17).

Example 1

However, more selectivity can be achieved with selective modifications of Polymyxin B as described in the following examples:

Example 2

Preparation of (18), (19), and (20), Compounds of Formula II Via Scheme B

The procedure is described in Bioorg. Med. Chem. Lett. 1998, 8, 3391-3396 and is as follows:

In the first step, polymyxin B monohydrochloride (50.0 mmol) is prepared as a homogeneous solution by dissolving the free base (1) in methanol containing AcOH (200 mmol) and HCl (50.0 mmol). In the second step, polymyxin B monohydrochloride (34.0 mmol) is reacted with di-tert-butyl dicarbonate (119.1 mmol) to afford the penta-BOC-derivative (18), and the tetra-BOC-derivatives (19) and (20), which are separated and purified by reverse phase HPLC.

Example 3

Preparation of (21), a Compound of Formula III Via Scheme C

Azelaic acid (2) (20 mmol) is dissolved in DMF and treated sequentially with hydroxybenzotriazole (50 mmol), diisopropylethyl amine (40 mmol) and PyBOP (40 mmol). After stirring for 15 minutes at room temperature, the activated diacid is treated with compound (19) (40 mmol), prepared as described in Example 2, and the coupling reaction is stirred overnight at room temperature. Volatiles are removed under vacuum and the crude is fractionated by reverse-phase HPLC to afford the title product after lyopholization of the appropriate fractions.

The product (10 mmol) is dissolved in CH₂Cl₂. A solution of 10% trifluoroacetic acid in CH₂Cl₂ is added and the reaction is stirred for 1 hour at room temperature. The solvent is then removed in vacuo to provide the desired material as the TFA salt. The desired material is then purified from this mixture using HPLC to afford the title product.

Example 4

Preparation of (23), a Compound of Formula IV Via Scheme D

A solution of compound (20) (1.0 mmol), prepared as described in Example 2, in 10 mL 0.1 M N-ethylmorpholineacetic acid buffer, pH 8.5 is treated with solid dimethyl adipimidate (22) (0.50 mmol). The resulting mixture is stirred at room temperature for 2 h, and then fractionated by reverse-phase HPLC.

The product (0.5 mmol) is dissolved in CH₂Cl₂. A solution of 10% trifluoroacetic acid in CH₂Cl₂ is added and the reaction is stirred for 1 hour at room temperature. The solvent is then removed in vacuo to provide the desired material as the TFA salt. The desired material is then purified from this mixture using HPLC to afford the title product.

Example 5

Preparation of (26), a Compound of Formula V Via Scheme E

Pentanedioic acid (24) (40 mmol) is dissolved in DMF and treated sequentially with hydroxybenzotriazole (50 mmol), diisopropylethyl amine (40 mmol) and PyBOP (40 mmol). After stirring for 15 minutes at room temperature, the activated acid is treated with compound (20) (40 mmol), prepared as described in Example 2, and the coupling reaction is stirred overnight at room temperature. Volatiles are removed under vacuum and the crude is fractionated by reverse-phase HPLC to afford the desired product (25) after lyopholization of the appropriate fractions.

The above product (25) (20 mmol) and compound 19 (Example 2) are dissolved in DMF and treated sequentially with hydroxybenzotriazole (25 mmol), diisopropylethyl amine (20 mmol) and PyBOP (20 mmol). The coupling reaction is stirred overnight at room temperature. Volatiles are removed under vacuum and the crude is fractionated by reverse-phase HPLC to afford the title product after lyopholization of the appropriate fractions.

The product (10 mmol) is dissolved in CH₂Cl₂. A solution of 10% trifluoroacetic acid in CH₂Cl₂ is added and the reaction is stirred for 1 hour at room temperature. The solvent is then removed in vacuo to provide the desired material as the TFA salt. The desired material is then purified from this mixture using HPLC to afford the title product.

Example 5

Example 6

Preparation of (28), a Compound of Formula VI

1,3,5-Benzenetricarboxylic acid (27) (10 mmol) is dissolved in DMF and treated sequentially with hydroxybenzotriazole (50 mmol), diisopropylethyl amine (40 mmol) and PyBOP (40 mmol). After stirring for 15 minutes at room temperature, the activated acid is treated with compound (19) (30 mmol), prepared as described in Example 2, and the coupling reaction is stirred overnight at room temperature. Volatiles are removed under vacuum and the crude is fractionated by reverse-phase HPLC to affords the title product after lyopholization of the appropriate fractions.

The product (10 mmol) is dissolved in CH₂Cl₂. A solution of 10% trifluoroacetic acid in CH₂Cl₂ is added and the reaction is stirred for 1 hour at room temperature. The solvent is then removed in vacuo to provide the desired material as the TFA salt. The desired material is then purified from this mixture using HPLC to afford the title product.

Note: Higher-order multivalomers can be synthesized using the same method but employing linkers containing multiple halogens.

Example 6

Example 7

Preparation of (29), a Compound of Formula VII

1,3,5-Benzenetricarboxylic acid (27) (10 mmol) is dissolved in DMF and treated sequentially with hydroxybenzotriazole (50 mmol), diisopropylethyl amine (40 mmol) and PyBOP (40 mmol). After stirring for 15 minutes at room temperature, the activated acid is treated with compound (20) (30 mmol), prepared as described in Example 2, and the coupling reaction is stirred overnight at room temperature. Volatiles are removed under vacuum and the crude is fractionated by reverse-phase HPLC to afford the title product after lyopholization of the appropriate fractions.

The product (10 mmol) is dissolved in CH₂Cl₂. A solution of 10% trifluoroacetic acid in CH₂Cl₂ is added and the reaction is stirred for 1 hour at room temperature. The solvent is then removed in vacuo to provide the desired material as the TFA salt. The desired material is then purified from this mixture using HPLC to afford the title product.

Note: Higher-order multivalomers can be synthesized using the same method but employing linkers containing multiple halogens.

Example 7 

What is claimed is:
 1. A compound of formula II: L′—X′—L′ wherein each L′ is independently a ligand comprising a polymyxin antibiotic and X′ is a linker attached on a γ-amino group on a DAB subunit of each ligand and having the formula: —X^(a)—Z—(Y^(a)—Z)_(m)—Y^(b)—Z—X^(a)— wherein m is an integer of from 0 to 20; X^(a) at each separate occurrence is selected from the group consisting of —O—, —S—, —NR—, —C(O)—, —C(O)O—, —C(O)NR—, —C(S)O—, —C(S)NR— or a covalent bond where R is as defined below; Z is at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond; Y^(a) and Y^(b) at each separate occurrence are selected from the group consisting of —C(O)NR′—, —NR′C(O)—, —NR′C(O)NR′—, —C(═NR′)—NR′—, —NR′—C(═NR′)—, —NR′—C(O)—O—, —P(O)(OR′)—O—, —S(O)_(n)CR′R″—, —S(O)_(n)—NR′—, —S—S— and a covalent bond; where n is 0, 1 or 2; and R, R′ and R″ at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic; or a pharmaceutically-acceptable salt thereof.
 2. A compound of claim 1 wherein each ligand is independently selected from the group consisting of polymyxin A, polymyxin B, polymyxin D1, polymyxin E1 and polymyxin E2.
 3. A compound as claimed in claim 2, wherein each ligand is polymyxin B, wherein the polymyxin B is independently selected from the group consisting of polymyxin B1 and polymyxin B2.
 4. A compound as claimed in claim 3, which is a bis(DAB₁, DAB₁-polymyxin B) dimer.
 5. A compound as claimed in claim 3, which is a bis(DAB₉, DAB₉-polymyxin B) dimer.
 6. A compound as claimed in claim 3, which is a bis(DAB₁, DAB₉-polymyxin B) dimer.
 7. A compound as claimed in claim 3, which is selected from:

wherein

represents polymyxin B.
 8. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a compound as claimed in claim
 1. 9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a compound as claimed in claim
 7. 